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Cells sense and respond to various mechanical forces from the extracellular matrix primarily by modulating the actin cytoskeleton. Mechanical forces can be translated into biochemical signals in a process called mechanotransduction. Yes-associated protein (YAP) is an effector of Hippo signaling and a mediator of mechanotransduction, but how mechanical forces regulate Hippo signaling is still an open question. We propose that retinoic acid-induced protein 14 (RAI14) responds to mechanical forces and regulates Hippo signaling. RAI14 positively regulates the activity of YAP. RAI14 interacts with NF2, a key component of the Hippo pathway, and the interaction occurs on filamentous actin. When mechanical forces are kept low in cells, NF2 dissociates from RAI14 and filamentous actin, resulting in increased interactions with LATS1 and activation of the Hippo pathway. Clinical data show that tissue stiffness and expression of RAI14 and YAP are upregulated in tumor tissues and that RAI14 is strongly associated with adverse outcome in patients with gastric cancer. Our data suggest that RAI14 links mechanotransduction with Hippo signaling and mediates Hippo-related biological functions such as cancer progression.
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Regulation of microtubule dynamics is required to properly control various steps of neurodevelopment. In this study, we identified granule cell antiserum-positive 14 (Gcap14) as a microtubule plus-end-tracking protein and as a regulator of microtubule dynamics during neurodevelopment. Gcap14 knockout mice exhibited impaired cortical lamination. Gcap14 deficiency resulted in defective neuronal migration. Moreover, nuclear distribution element nudE-like 1 (Ndel1), an interacting partner of Gcap14, effectively corrected the downregulation of microtubule dynamics and the defects in neuronal migration caused by Gcap14 deficiency. Finally, we found that the Gcap14-Ndel1 complex participates in the functional link between microtubule and actin filament, thereby regulating their crosstalks in the growth cones of cortical neurons. Taken together, we propose that the Gcap14-Ndel1 complex is fundamental for cytoskeletal remodeling during neurodevelopmental processes such as neuronal processes elongation and neuronal migration.
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Actinas , Proteínas Asociadas a Microtúbulos , Neuronas , Animales , Ratones , Actinas/metabolismo , Movimiento Celular/fisiología , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Neuritas/metabolismo , Neuronas/metabolismoRESUMEN
The endoplasmic reticulum (ER) and mitochondria form a unique subcellular compartment called mitochondria-associated ER membranes (MAMs). Disruption of MAMs impairs Ca2+ homeostasis, triggering pleiotropic effects in the neuronal system. Genome-wide kinase-MAM interactome screening identifies casein kinase 2 alpha 1 (CK2A1) as a regulator of composition and Ca2+ transport of MAMs. CK2A1-mediated phosphorylation of PACS2 at Ser207/208/213 facilitates MAM localization of the CK2A1-PACS2-PKD2 complex, regulating PKD2-dependent mitochondrial Ca2+ influx. We further reveal that mutations of PACS2 (E209K and E211K) associated with developmental and epileptic encephalopathy-66 (DEE66) impair MAM integrity through the disturbance of PACS2 phosphorylation at Ser207/208/213. This, in turn, causes the reduction of mitochondrial Ca2+ uptake and the dramatic increase of the cytosolic Ca2+ level, thereby, inducing neurotransmitter release at the axon boutons of glutamatergic neurons. In conclusion, our findings suggest a molecular mechanism that MAM alterations induced by pathological PACS2 mutations modulate Ca2+-dependent neurotransmitter release.
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Retículo Endoplásmico , Mitocondrias , Mitocondrias/metabolismo , Retículo Endoplásmico/metabolismo , Fosforilación , Neurotransmisores/metabolismoRESUMEN
Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Here, we present a reliable, and simply reproducible process for constructing user-controlled long rounded extracellular matrix (ECM)-embedded vascular microlumens on-chip for endothelization and co-culture with stromal cells obtained from human lung. We demonstrate the critical impact of microchannel cross-sectional geometry and length on uniform distribution and magnitude of vascular wall shear stress, which is key when emulating in vivo-observed blood flow biomechanics in health and disease. In addition, we provide an optimization protocol for multicellular culture and functional validation of the system. Moreover, we show the ability to finely tune rheology of the three-dimensional natural matrix surrounding the vascular microchannel to match pathophysiological stiffness. In summary, we provide the scientific community with a matrix-embedded microvasculature on-chip populated with all-primary human-derived pulmonary endothelial cells and fibroblasts to recapitulate and interrogate lung parenchymal biology, physiological responses, vascular biomechanics, and disease biogenesis in vitro. Such a mix-and-match synthetic platform can be feasibly adapted to study blood vessels, matrix, and ECM-embedded cells in other organs and be cellularized with additional stromal cells.
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Although large-scale genome-wide association studies (GWAS) have identified an association between MAD1L1 (Mitotic Arrest Deficient-1 Like 1) and the pathology of schizophrenia, the molecular mechanisms underlying this association remain unclear. In the present study, we aimed to address these mechanisms by examining the role of MAD1 (the gene product of MAD1L1) in key neurodevelopmental processes in mice and human organoids. Our findings indicated that MAD1 is highly expressed during active cortical development and that MAD1 deficiency leads to impairments in neuronal migration and neurite outgrowth. We also observed that MAD1 is localized to the Golgi apparatus and regulates vesicular trafficking from the Golgi apparatus to the plasma membrane, which is required for the growth and polarity of migrating neurons. In this process, MAD1 physically interacts and collaborates with the kinesin-like protein KIFC3 (kinesin family member C3) to regulate the morphology of the Golgi apparatus and neuronal polarity, thereby ensuring proper neuronal migration and differentiation. Consequently, our findings indicate that MAD1 is an essential regulator of neuronal development and that alterations in MAD1 may underlie schizophrenia pathobiology.
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Neocórtex , Esquizofrenia , Animales , Humanos , Ratones , Proteínas de Ciclo Celular/genética , Estudio de Asociación del Genoma Completo , Cinesinas/genética , Cinesinas/metabolismo , Neocórtex/metabolismo , Neuronas/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismoRESUMEN
The mitochondria-associated membrane (MAM) has emerged as a cellular signaling hub regulating various cellular processes. However, its molecular components remain unclear owing to lack of reliable methods to purify the intact MAM proteome in a physiological context. Here, we introduce Contact-ID, a split-pair system of BioID with strong activity, for identification of the MAM proteome in live cells. Contact-ID specifically labeled proteins proximal to the contact sites of the endoplasmic reticulum (ER) and mitochondria, and thereby identified 115 MAM-specific proteins. The identified MAM proteins were largely annotated with the outer mitochondrial membrane (OMM) and ER membrane proteins with MAM-related functions: e.g., FKBP8, an OMM protein, facilitated MAM formation and local calcium transport at the MAM. Furthermore, the definitive identification of biotinylation sites revealed membrane topologies of 85 integral membrane proteins. Contact-ID revealed regulatory proteins for MAM formation and could be reliably utilized to profile the proteome at any organelle-membrane contact sites in live cells.
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Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Proteoma/análisis , Proteínas de Unión a Tacrolimus/metabolismo , Calcio/metabolismo , Humanos , Biogénesis de Organelos , Proteoma/metabolismo , Transducción de SeñalRESUMEN
FMRP is a multifunctional protein encoded by the Fragile X Messenger Ribonucleoprotein 1 gene (FMR1). The inactivation of the FMR1 gene results in fragile X syndrome (FXS), a serious neurodevelopmental disorder. FMRP deficiency causes abnormal neurite outgrowth, which is likely to lead to abnormal learning and memory capabilities. However, the mechanism of FMRP in modulating neuronal development remains unknown. We found that FMRP enhances the translation of 4EBP2, a neuron-specific form of 4EBPs that inactivates eIF4E by inhibiting the interaction between eIF4E and eIF4G. Depletion of 4EBP2 results in abnormal neurite outgrowth. Moreover, the impairment of neurite outgrowth upon FMRP depletion was overcome by the ectopic expression of 4EBP2. These results suggest that FMRP controls neuronal development by enhancing 4EBP2 expression at the translational level. In addition, treatment with 4EGI-1, a chemical that blocks eIF4E activity, restored neurite length in FMRP-depleted and 4EBP2-depleted cells. In conclusion, we discovered that 4EBP2 functions as a key downstream regulator of FMRP activity in neuronal development and that FMRP represses eIF4E activity by enhancing 4EBP2 translation.
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Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Humanos , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Neuronas/metabolismo , Síndrome del Cromosoma X Frágil/genética , Diferenciación Celular/genéticaRESUMEN
The glycoprotein Reelin maintains neuronal positioning and regulates neuronal plasticity in the adult brain. Reelin deficiency has been associated with neurological diseases. We recently showed that Reelin is depleted in mice with a targeted disruption of the Ndel1 gene in forebrain postnatal excitatory neurons (Ndel1 conditional knockout (CKO)). Ndel1 CKO mice exhibit fragmented microtubules in CA1 pyramidal neurons, profound deterioration of the CA1 hippocampus and a shortened lifespan (~10 weeks). Here we report that Ndel1 CKO mice (of both sexes) experience spatial learning and memory deficits that are associated with deregulation of neuronal cell adhesion, plasticity and neurotransmission genes, as assessed by genome-wide transcriptome analysis of the hippocampus. Importantly, a single injection of Reelin protein in the hippocampus of Ndel1 CKO mice improves spatial learning and memory function and this is correlated with reduced intrinsic hyperexcitability of CA1 pyramidal neurons, and normalized gene deregulation in the hippocampus. Strikingly, when treated with Reelin, Ndel1 CKO animals that die from an epileptic phenotype, live twice as long as nontreated, or vehicle-treated CKO animals. Thus, Reelin confers striking beneficial effects in the CA1 hippocampus, and at both behavioral and organismal levels.
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Región CA1 Hipocampal/patología , Proteínas Portadoras/genética , Longevidad/efectos de los fármacos , Proteína Reelina/farmacología , Animales , Región CA1 Hipocampal/efectos de los fármacos , Cognición/efectos de los fármacos , Femenino , Longevidad/genética , Masculino , Trastornos de la Memoria/genética , Ratones , Ratones Noqueados , Mutación , Aprendizaje Espacial/efectos de los fármacosRESUMEN
Perilipin 2 (PLIN2) is a major lipid droplet (LD)-associated protein that regulates intracellular lipid homeostasis and LD formation. Under lipid-deprived conditions, the LD-unbound (free) form of PLIN2 is eliminated in the cytosol by an as yet unknown ubiquitin (Ub)-proteasome pathway that is associated with the N-terminal or near N-terminal residues of the protein. Here, using HeLa, HEK293T, and HepG2 human cell lines, cycloheximide chase, in vivo ubiquitylation, split-Ub yeast two-hybrid, and chemical cross-linking-based reciprocal co-immunoprecipitation assays, we found that TEB4 (MARCH6), an E3 Ub ligase and recognition component of the Ac/N-end rule pathway, directly targets the N-terminal acetyl moiety of Nα-terminally acetylated PLIN2 for its polyubiquitylation and degradation by the 26S proteasome. We also show that the TEB4-mediated Ac/N-end rule pathway reduces intracellular LD accumulation by degrading PLIN2. Collectively, these findings identify PLIN2 as a substrate of the Ac/N-end rule pathway and indicate a previously unappreciated role of the Ac/N-end rule pathway in LD metabolism.
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Gotas Lipídicas/metabolismo , Perilipina-2/metabolismo , Proteolisis , Ubiquitinación , Acetilación , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Perilipina-2/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Dominios Proteicos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The dopaminergic system plays an essential role in various functions of the brain, including locomotion, memory, and reward, and the deregulation of dopaminergic signaling as a result of altered functionality of dopamine D2 receptor (DRD2) is implicated in multiple neurologic and psychiatric disorders. Tetraspanin-7 (TSPAN7) is expressed to variable degrees in different tissues, with the highest level in the brain, and multiple mutations in TSPAN7 have been implicated in intellectual disability. Here, we tested the hypothesis that TSPAN7 may be a binding partner of DRD2 that is involved in the regulation of its functional activity. Our results showed that TSPAN7 was associated with DRD2 and reduced its surface expression by enhancing DRD2 internalization. Immunocytochemical analysis revealed that TSPAN7 that resides in the plasma membrane and early and late endosomes promoted internalization of DRD2 and its localization to endosomal compartments of the endocytic pathway. Furthermore, we observed that TSPAN7 deficiency increased surface localization of DRD2 concurrent with the decrease of its endocytosis, regardless of dopamine treatment. Finally, TSPAN7 negatively affects DRD2-mediated signaling. These results disclosed a previously uncharacterized role of TSPAN7 in the regulation of the expression and functional activity of DRD2 by postendocytic trafficking.-Lee, S.-A., Suh, Y., Lee, S., Jeong, J., Kim, S. J., Kim, S. J., Park, S. K. Functional expression of dopamine D2 receptor is regulated by tetraspanin 7-mediated postendocytic trafficking.
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Proteínas del Tejido Nervioso/metabolismo , Receptores de Dopamina D2/metabolismo , Tetraspaninas/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas del Tejido Nervioso/genética , Isoformas de Proteínas , Transporte de Proteínas , Receptores de Dopamina D2/genética , Transducción de Señal , Tetraspaninas/genéticaRESUMEN
INTRODUCTION: Crotonaldehyde is an α,ß-unsaturated carbonyl compound that is a potent eye, respiratory, and skin irritant. Crotonaldehyde is a major constituent of tobacco smoke and its exposure can be quantified using its urinary metabolite N-acetyl-S-(3-hydroxypropyl-1-methyl)-L-cysteine (HPMM). A large-scale biomonitoring study is needed to determine HPMM levels, as a measure of crotonaldehyde exposure, in the general U.S. MATERIALS AND METHODS: Urine samples were obtained as part of the National Health and Nutrition Examination Survey 2005-2006 and 2011-2012 from participants who were at least six-years-old (N = 4692). Samples were analyzed for HPMM using ultra performance liquid chromatography - tandem mass spectrometry. Exclusive tobacco smokers were distinguished from non- tobacco users through a combination of self-reporting and serum cotinine data. RESULTS: Detection rate of HPMM among eligible samples was 99.9%. Sample-weighted, median urinary HPMM levels for smokers and non-users were 1.61 and 0.313â¯mg/g creatinine, respectively. Multivariable regression analysis among smokers showed that HPMM was positively associated with serum cotinine, after controlling for survey year, urinary creatinine, age, sex, race, poverty level, body mass index, pre-exam fasting time, and food intake. Other significant predictors of urinary HPMM include sex (female > male), age (children > non-user adults), race (non-Hispanic Blacks < non-Hispanic Whites). CONCLUSIONS: This study characterizes U.S. population exposure to crotonaldehyde and confirms that tobacco smoke is a major exposure source. Urinary HPMM levels were significantly higher among exclusive combusted tobacco users compared to non-users, and serum cotinine and cigarettes per day were significant predictors of increased urinary HPMM. This study also found that sex, age, ethnicity, pre-exam fasting time, and fruit consumption are related to urinary HPMM levels.
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Cotinina , Fumadores , Contaminación por Humo de Tabaco , Adolescente , Adulto , Anciano , Aldehídos , Niño , Cotinina/orina , Exposición a Riesgos Ambientales , Femenino , Humanos , Masculino , Persona de Mediana Edad , No Fumadores , Encuestas Nutricionales , Estados Unidos , Adulto JovenRESUMEN
UNLABELLED: How the integrity of laminar structures in the postnatal brain is maintained impacts neuronal functions. Ndel1, the mammalian homolog of NuDE from the filamentous fungus Aspergillus nidulans, is an atypical microtubule (MT)-associated protein that was initially investigated in the contexts of neurogenesis and neuronal migration. Constitutive knock-out mice for Ndel1 are embryonic lethal, thereby necessitating the creation a conditional knock-out to probe the roles of Ndel1 in postnatal brains. Here we report that CA1 pyramidal neurons from mice postnatally lacking Ndel1 (Ndel1 conditional knock-out) exhibit fragmented MTs, dendritic/synaptic pathologies, are intrinsically hyperexcitable and undergo dispersion independently of neuronal migration defect. Secondary to the pyramidal cell changes is the decreased inhibitory drive onto pyramidal cells from interneurons. Levels of the glycoprotein Reelin that regulates MTs, neuronal plasticity, and cell compaction are significantly reduced in hippocampus of mutant mice. Strikingly, a single injection of Reelin into the hippocampus of Ndel1 conditional knock-out mice ameliorates ultrastructural, cellular, morphological, and anatomical CA1 defects. Thus, Ndel1 and Reelin contribute to maintain postnatal CA1 integrity. SIGNIFICANCE STATEMENT: The significance of this study rests in the elucidation of a role for Nde1l and Reelin in postnatal CA1 integrity using a new conditional knock-out mouse model for the cytoskeletal protein Ndel1, one that circumvents the defects associated with neuronal migration and embryonic lethality. Our study serves as a basis for understanding the mechanisms underlying postnatal hippocampal maintenance and function, and the significance of decreased levels of Ndel1 and Reelin observed in patients with neurological disorders.
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Región CA1 Hipocampal/crecimiento & desarrollo , Región CA1 Hipocampal/metabolismo , Moléculas de Adhesión Celular Neuronal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas del Tejido Nervioso/metabolismo , Serina Endopeptidasas/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Región CA1 Hipocampal/ultraestructura , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/farmacología , Proteínas de Ciclo Celular/genética , Dendritas/metabolismo , Dendritas/ultraestructura , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/genética , Glutamato Descarboxilasa/metabolismo , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/farmacología , Proteína Reelina , Serina Endopeptidasas/genética , Serina Endopeptidasas/farmacología , Tinción con Nitrato de Plata , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
The human ortholog of the targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a cytoskeletal protein that plays a major role in spindle assembly and is required for mitosis. During spindle morphogenesis, TPX2 cooperates with Aurora A kinase and Eg5 kinesin to regulate microtubule organization. TPX2 displays over 40 putative phosphorylation sites identified from various high-throughput proteomic screenings. In this study, we characterize the phosphorylation of threonine 72 (Thr(72)) in human TPX2, a residue highly conserved across species. We find that Cdk1/2 phosphorylate TPX2 in vitro and in vivo. Using homemade antibodies specific for TPX2 phosphorylated at Thr(72), we show that this phosphorylation is cell cycle-dependent and peaks at M phase. Endogenous TPX2 phosphorylated at Thr(72) does not associate with the mitotic spindle. Furthermore, ectopic GFP-TPX2 T72A preferentially concentrates on the spindle, whereas GFP-TPX2 WT distributes to both spindle and cytosol. The T72A mutant also increases the proportion of cells with multipolar spindles phenotype. This effect is associated with increased Aurora A activity and abnormally elongated spindles, indicative of higher Eg5 activity. In summary, we propose that phosphorylation of Thr(72) regulates TPX2 localization and impacts spindle assembly via Aurora A and Eg5.
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Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Huso Acromático , Treonina/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/química , Cartilla de ADN , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Nucleares/química , Fosfoproteínas/química , Fosforilación , Treonina/química , Xenopus , Proteínas de Xenopus/químicaRESUMEN
Dysbindin and DISC1 are schizophrenia susceptibility factors playing roles in neuronal development. Here we show that the physical interaction between dysbindin and DISC1 is critical for the stability of dysbindin and for the process of neurite outgrowth. We found that DISC1 forms a complex with dysbindin and increases its stability in association with a reduction in ubiquitylation. Furthermore, knockdown of DISC1 or expression of a deletion mutant, DISC1 lacking amino acid residues 403-504 of DISC1 (DISC1(Δ403-504)), effectively decreased levels of endogenous dysbindin. Finally, the neurite outgrowth defect induced by knockdown of DISC1 was partially reversed by coexpression of dysbindin. Taken together, these results indicate that dysbindin and DISC1 form a physiologically functional complex that is essential for normal neurite outgrowth.
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Proteínas Asociadas a la Distrofina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Células COS , Células Cultivadas , Corteza Cerebral/citología , Chlorocebus aethiops , Disbindina , Proteínas Asociadas a la Distrofina/química , Células HEK293 , Humanos , Ratones , Proteínas del Tejido Nervioso/química , Neuritas/metabolismo , Neuritas/patología , Neuronas/metabolismo , Neuronas/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , UbiquitinaRESUMEN
Circadian rhythm is a biological rhythm governing physiology and behavior with a period of â¼24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.
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Proteínas CLOCK/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Activación Transcripcional/fisiología , Animales , Proteínas CLOCK/genética , Quinasa 5 Dependiente de la Ciclina/genética , Células HEK293 , Humanos , Ratones , Células 3T3 NIH , Fosforilación/fisiología , Estabilidad Proteica , Transporte de Proteínas/fisiología , Treonina/genética , Treonina/metabolismoRESUMEN
Identifying proteins at organelle contact sites, such as mitochondria-associated endoplasmic reticulum membranes (MAM), is essential for understanding vital cellular processes, yet challenging due to their dynamic nature. Here we report "OrthoID", a proteomic method utilizing engineered enzymes, TurboID and APEX2, for the biotinylation (Bt) and adamantylation (Ad) of proteins close to the mitochondria and endoplasmic reticulum (ER), respectively, in conjunction with high-affinity binding pairs, streptavidin-biotin (SA-Bt) and cucurbit[7]uril-adamantane (CB[7]-Ad), for selective orthogonal enrichment of Bt- and Ad-labeled proteins. This approach effectively identifies protein candidates associated with the ER-mitochondria contact, including LRC59, whose roles at the contact site were-to the best of our knowledge-previously unknown, and tracks multiple protein sets undergoing structural and locational changes at MAM during mitophagy. These findings demonstrate that OrthoID could be a powerful proteomics tool for the identification and analysis of spatiotemporal proteins at organelle contact sites and revealing their dynamic behaviors in vital cellular processes.
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Proteoma , Proteómica , Proteoma/metabolismo , Proteómica/métodos , Membranas Mitocondriales/metabolismo , Mitocondrias/metabolismo , Retículo Endoplásmico/metabolismoRESUMEN
Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.
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Eritropoyetina/fisiología , Hígado/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Hematócrito , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Ratones Transgénicos , Modelos Animales , Policitemia/fisiopatología , Receptores de Factores de Crecimiento Endotelial Vascular/fisiología , Vasos Retinianos/fisiologíaRESUMEN
Disrupted-in-schizophrenia 1 (DISC1) has emerged as a schizophrenia-susceptibility gene affecting various neuronal functions. In this study, we characterized Mitofilin, a mitochondrial inner membrane protein, as a mediator of the mitochondrial function of DISC1. A fraction of DISC1 was localized to the inside of mitochondria and directly interacts with Mitofilin. A reduction in DISC1 function induced mitochondrial dysfunction, evidenced by decreased mitochondrial NADH dehydrogenase activities, reduced cellular ATP contents, and perturbed mitochondrial Ca(2+) dynamics. In addition, deficiencies in DISC1 and Mitofilin induced a reduction in mitochondrial monoamine oxidase-A activity. The mitochondrial dysfunctions evoked by the deficiency of DISC1 were partially phenocopied by an overexpression of truncated DISC1 that is associated with schizophrenia in human. DISC1 deficiencies induced the ubiquitination of Mitofilin, suggesting that DISC1 is critical for the stability of Mitofilin. Finally, the mitochondrial dysfunction induced by DISC1 deficiency was partially reversed by coexpression of Mitofilin, confirming a functional link between DISC1 and Mitofilin for the normal mitochondrial function. According to these results, we propose that DISC1 plays essential roles for mitochondrial function in collaboration with a mitochondrial interacting partner, Mitofilin.
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Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Esquizofrenia/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Proteínas Mitocondriales/genética , Monoaminooxidasa/metabolismo , Proteínas Musculares/genética , NADH Deshidrogenasa/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Linear nevus sebaceous syndrome (LNSS) is a neurocutaneous disorder caused by somatic gain-of-function mutations in KRAS or HRAS. LNSS brains have neurodevelopmental defects, including cerebral defects and epilepsy; however, its pathological mechanism and potentials for treatment are largely unclear. We show that introduction of KRASG12V in the developing mouse cortex results in subcortical nodular heterotopia and enhanced excitability, recapitulating major pathological manifestations of LNSS. Moreover, we show that decreased firing frequency of inhibitory neurons without KRASG12V expression leads to disrupted excitation and inhibition balance. Transcriptional profiling after destabilization domain-mediated clearance of KRASG12V in human neural progenitors and differentiating neurons identifies reversible functional networks underlying LNSS. Neurons expressing KRASG12V show molecular changes associated with delayed neuronal maturation, most of which are restored by KRASG12V clearance. These findings provide insights into the molecular networks underlying the reversibility of some of the neuropathologies observed in LNSS caused by dysregulation of the RAS pathway.
Asunto(s)
Epilepsia , Nevo Sebáceo de Jadassohn , Ratones , Animales , Humanos , Proteínas Proto-Oncogénicas p21(ras)/genética , Nevo Sebáceo de Jadassohn/genética , Nevo Sebáceo de Jadassohn/patología , Neuropatología , Mutación/genéticaRESUMEN
The adverse effects of silver nanoparticles (AgNPs) have been studied in various models. However, there has been discordance between molecular responses across the literature, attributed to methodological biases and the physicochemical variability of AgNPs. In this study, a gene pathway meta-analysis was conducted to identify convergent and divergent key events (KEs) associated with AgNPs and explore common patterns of these KEs across species. We performed a cross-species analysis of transcriptomic data from multiple studies involving various AgNPs exposure. Pathway enrichment analysis revealed a set of pathways linked to oxidative stress, apoptosis, and metabolite and lipid metabolism, which are considered potentially conserved KEs across species. Subsequently, experiments confirmed that oxidative stress responses could be early KEs in both Caenorhabditis elegans and HepG2 cells. Moreover, AgNPs preferentially impaired the mitochondria, as evidenced by mitochondrial fragmentation and dysfunction. Furthermore, disruption of amino acids, nucleotides, sulfur compounds, glycerolipids, and glycerophospholipids metabolism were in good agreement with gene pathway shreds of evidence. Our findings imply that, although there may be organism-specific responses, potentially conserved events could exist regardless of species and physicochemical factors. These results provide valuable insights into the development of adverse outcome pathways of AgNPs across species and the regulatory toxicity of AgNPs.