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1.
Nat Struct Mol Biol ; 29(10): 1024-1036, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36220893

RESUMEN

The regular functioning of the nucleolus and nucleus-mitochondria crosstalk are considered unrelated processes, yet cytochrome c (Cc) migrates to the nucleus and even the nucleolus under stress conditions. Nucleolar liquid-liquid phase separation usually serves the cell as a fast, smart mechanism to control the spatial localization and trafficking of nuclear proteins. Actually, the alternative reading frame (ARF), a tumor suppressor protein sequestered by nucleophosmin (NPM) in the nucleoli, is shifted out from NPM upon DNA damage. DNA damage also triggers early translocation of respiratory Cc to nucleus before cytoplasmic caspase activation. Here, we show that Cc can bind to nucleolar NPM by triggering an extended-to-compact conformational change, driving ARF release. Such a NPM-Cc nucleolar interaction can be extended to a general mechanism for DNA damage in which the lysine-rich regions of Cc-rather than the canonical, arginine-rich stretches of membrane-less organelle components-controls the trafficking and availability of nucleolar proteins.


Asunto(s)
Citocromos c , Nucleofosmina , Arginina , Caspasas , Lisina , Mitocondrias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor
2.
Redox Biol ; 43: 101967, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-33882408

RESUMEN

Repair of injured DNA relies on nucleosome dismantling by histone chaperones and de-phosphorylation events carried out by Protein Phosphatase 2A (PP2A). Typical histone chaperones are the Acidic leucine-rich Nuclear Phosphoprotein 32 family (ANP32) members, e.g. ANP32A, which is also a well-known PP2A inhibitor (a.k.a. I1PP2A). Here we report the novel interaction between the endogenous family member B-so-called ANP32B-and endogenous cytochrome c in cells undergoing camptothecin-induced DNA damage. Soon after DNA lesions but prior to caspase cascade activation, the hemeprotein translocates to the nucleus to target the Low Complexity Acidic Region (LCAR) of ANP32B; in a similar way, our group recently reported that the hemeprotein targets the acidic domain of SET/Template Activating Factor-Iß (SET/TAF-Iß), which is another histone chaperone and PP2A inhibitor (a.k.a. I2PP2A). The nucleosome assembly activity of ANP32B is indeed unaffected by cytochrome c binding. Like ANP32A, ANP32B inhibits PP2A activity and is thus herein referred to as I3PP2A. Our data demonstrates that ANP32B-dependent inhibition of PP2A is regulated by respiratory cytochrome c, which induces long-distance allosteric changes in the structured N-terminal domain of ANP32B upon binding to the C-terminal LCAR. In agreement with the reported role of PP2A in the DNA damage response, we propose a model wherein cytochrome c is translocated from the mitochondria into the nucleus upon DNA damage to modulate PP2A activity via its interaction with ANP32B.


Asunto(s)
Citocromos c , Chaperonas de Histonas , Núcleo Celular , Daño del ADN , Proteína Fosfatasa 2
3.
Elife ; 102021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33491649

RESUMEN

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease with impaired organ growth and increased tumor formation. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterize these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We find that an atypical de novo assembly pathway can generate Cenpas that act as microtubule-organizing centers (MTOCs), including in Mulibrey patient cells. Correlative light electron microscopy reveals that Cenpas are centriole-related or electron-dense structures with stripes. TRIM37 regulates the stability and solubility of Centrobin, which accumulates in elongated entities resembling the striped electron dense structures upon TRIM37 depletion. Furthermore, Cenpas formation upon TRIM37 depletion requires PLK4, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents Cenpas formation, which would otherwise threaten genome integrity.


Asunto(s)
Proteínas de Ciclo Celular/genética , Centro Organizador de los Microtúbulos/metabolismo , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Centriolos/metabolismo , Células HeLa , Humanos , Enanismo Mulibrey/genética , Enanismo Mulibrey/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo
4.
Nat Commun ; 10(1): 2864, 2019 06 28.
Artículo en Inglés | MEDLINE | ID: mdl-31253807

RESUMEN

The T cell immune synapse is a site of intense vesicular trafficking. Here we show that the golgin GMAP210, known to capture vesicles and organize membrane traffic at the Golgi, is involved in the vesicular transport of LAT to the immune synapse. Upon activation, more GMAP210 interact with LAT-containing vesicles and go together with LAT to the immune synapse. Regulating LAT recruitment and LAT-dependent signaling, GMAP210 controls T cell activation. Using a rerouting and capture assay, we show that GMAP210 captures VAMP7-decorated vesicles. Overexpressing different domains of GMAP210, we also show that GMAP210 allows their specific delivery to the immune synapse by tethering LAT-vesicles to the Golgi. Finally, in a model of ectopic expression of LAT in ciliated cells, we show that GMAP210 tethering activity controls the delivery of LAT to the cilium. Hence, our results reveal a function for the golgin GMAP210 conveying specific vesicles to the immune synapse.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aparato de Golgi/fisiología , Leucocitos Mononucleares/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Vesículas Transportadoras/fisiología , Línea Celular , Proteínas del Citoesqueleto , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Proteínas Nucleares/genética , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de Señal , Linfocitos T/fisiología
5.
Rev. bras. psiquiatr ; 41(1): 70-81, Jan.-Mar. 2019. tab, graf
Artículo en Inglés | LILACS | ID: biblio-985353

RESUMEN

Objective: Noninvasive brain stimulation (NIBS) techniques, such as transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS), are increasingly being used to treat mental disorders, particularly major depression. The aim of this comprehensive review is to summarize the main advances, limitations, and perspectives of the field. Methods: We searched PubMed and other databases from inception to July 2017 for articles, particularly systematic reviews and meta-analyses, evaluating the use of NIBS in psychiatric disorders. Results: We reviewed the mechanisms of action, safety, tolerability, efficacy, and relevant clinical parameters of NIBS. Repetitive TMS is already an established technique for the treatment of depression, and there is theoretically room for further methodological development towards a high-end therapeutic intervention. In contrast, tDCS is a technically easier method and therefore potentially suitable for wider clinical use. However the evidence of its antidepressant efficacy is less sound, and a recent study found tDCS to be inferior to antidepressant pharmacotherapy. Clinical trials using rTMS for other mental disorders produced mixed findings, whereas tDCS use has not been sufficiently appraised. Conclusion: The most promising results of NIBS have been obtained for depression. These techniques excel in safety and tolerability, although their efficacy still warrants improvement.


Asunto(s)
Humanos , Estimulación Magnética Transcraneal/métodos , Estimulación Transcraneal de Corriente Directa/métodos , Trastornos Mentales/terapia , Ensayos Clínicos como Asunto , Medicina Basada en la Evidencia
6.
Braz J Psychiatry ; 41(1): 70-81, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30328957

RESUMEN

OBJECTIVE: Noninvasive brain stimulation (NIBS) techniques, such as transcranial direct current stimulation (tDCS) and repetitive transcranial magnetic stimulation (rTMS), are increasingly being used to treat mental disorders, particularly major depression. The aim of this comprehensive review is to summarize the main advances, limitations, and perspectives of the field. METHODS: We searched PubMed and other databases from inception to July 2017 for articles, particularly systematic reviews and meta-analyses, evaluating the use of NIBS in psychiatric disorders. RESULTS: We reviewed the mechanisms of action, safety, tolerability, efficacy, and relevant clinical parameters of NIBS. Repetitive TMS is already an established technique for the treatment of depression, and there is theoretically room for further methodological development towards a high-end therapeutic intervention. In contrast, tDCS is a technically easier method and therefore potentially suitable for wider clinical use. However the evidence of its antidepressant efficacy is less sound, and a recent study found tDCS to be inferior to antidepressant pharmacotherapy. Clinical trials using rTMS for other mental disorders produced mixed findings, whereas tDCS use has not been sufficiently appraised. CONCLUSION: The most promising results of NIBS have been obtained for depression. These techniques excel in safety and tolerability, although their efficacy still warrants improvement.


Asunto(s)
Trastornos Mentales/terapia , Estimulación Transcraneal de Corriente Directa/métodos , Estimulación Magnética Transcraneal/métodos , Ensayos Clínicos como Asunto , Medicina Basada en la Evidencia , Humanos
7.
iScience ; 4: 44-63, 2018 Jun 29.
Artículo en Inglés | MEDLINE | ID: mdl-30240752

RESUMEN

Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR-/-) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR-/- mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/ß-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR-/- liver. Consistently, PI3K, ERK, and Wnt/ß-catenin inhibition partially rescued polyploidy in AhR-/- mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.

8.
EMBO Rep ; 19(11)2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30224411

RESUMEN

Here, we address the regulation of microtubule nucleation during interphase by genetically ablating one, or two, of three major mammalian γ-TuRC-binding factors namely pericentrin, CDK5Rap2, and AKAP450. Unexpectedly, we find that while all of them participate in microtubule nucleation at the Golgi apparatus, they only modestly contribute at the centrosome where CEP192 has a more predominant function. We also show that inhibiting microtubule nucleation at the Golgi does not affect centrosomal activity, whereas manipulating the number of centrosomes with centrinone modifies microtubule nucleation activity of the Golgi apparatus. In centrosome-free cells, inhibition of Golgi-based microtubule nucleation triggers pericentrin-dependent formation of cytoplasmic-nucleating structures. Further depletion of pericentrin under these conditions leads to the generation of individual microtubules in a γ-tubulin-dependent manner. In all cases, a conspicuous MT network forms. Strikingly, centrosome loss increases microtubule number independently of where they were growing from. Our results lead to an unexpected view of the interphase centrosome that would control microtubule network organization not only by nucleating microtubules, but also by modulating the activity of alternative microtubule-organizing centers.


Asunto(s)
Centrosoma/metabolismo , Interfase/fisiología , Microtúbulos/metabolismo , Proteínas de Anclaje a la Quinasa A/genética , Antígenos/genética , Sistemas CRISPR-Cas , Proteínas de Ciclo Celular , Línea Celular , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas del Citoesqueleto/genética , Técnicas de Inactivación de Genes , Aparato de Golgi/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Tubulina (Proteína)/metabolismo
9.
Microbiology (Reading) ; 163(2): 253-265, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28270274

RESUMEN

The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.


Asunto(s)
Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , ADN Helicasas/genética , Regulación Bacteriana de la Expresión Génica/genética , ARN sin Sentido/genética , Salmonella typhi/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Transcripción Genética/genética , Activación Transcripcional/genética , Regulación hacia Arriba/genética
10.
Neuropsychopharmacology ; 42(13): 2593-2601, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28145409

RESUMEN

Bipolar depression (BD) is a highly prevalent condition with limited therapeutic options. Deep (H1-coil) transcranial magnetic stimulation (dTMS) is a novel TMS modality with established efficacy for unipolar depression. We conducted a randomized sham-controlled trial to evaluate the efficacy and safety of dTMS in treatment-resistant BD patients. Patients received 20 sessions of active or sham dTMS over the left dorsolateral prefrontal cortex (H1-coil, 55 18 Hz 2 s 120% MT trains). The primary outcome was changes in the 17-item Hamilton Depression Rating Scale (HDRS-17) from baseline to endpoint (week 4). Secondary outcomes were changes from baseline to the end of the follow-up phase (week 8), and response and remission rates. Safety was assessed using a dTMS adverse effects questionnaire and the Young Mania Rating Scale to assess treatment-emergent mania switch (TEMS). Out of 50 patients, 43 finished the trial. There were 2 and 5 dropouts in the sham and active groups, respectively. Active dTMS was superior to sham at end point (difference favoring dTMS=4.88; 95% CI 0.43 to 9.32, p=0.03) but not at follow-up. There was also a trend for greater response rates in the active (48%) vs sham (24%) groups (OR=2.92; 95% CI=0.87 to 9.78, p=0.08). Remission rates were not statistically different. No TEMS episodes were observed. Deep TMS is a potentially effective and well-tolerated add-on therapy in resistant bipolar depressed patients receiving adequate pharmacotherapy.


Asunto(s)
Trastorno Bipolar/terapia , Estimulación Magnética Transcraneal , Adulto , Antidepresivos/uso terapéutico , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Masculino , Pacientes Desistentes del Tratamiento , Corteza Prefrontal , Escalas de Valoración Psiquiátrica , Inducción de Remisión , Factores de Tiempo , Estimulación Magnética Transcraneal/efectos adversos , Estimulación Magnética Transcraneal/métodos , Resultado del Tratamiento
11.
PLoS Biol ; 13(3): e1002087, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25764135

RESUMEN

Epithelial morphogenesis involves a dramatic reorganisation of the microtubule cytoskeleton. How this complex process is controlled at the molecular level is still largely unknown. Here, we report that the centrosomal microtubule (MT)-binding protein CAP350 localises at adherens junctions in epithelial cells. By two-hybrid screening, we identified a direct interaction of CAP350 with the adhesion protein α-catenin that was further confirmed by co-immunoprecipitation experiments. Block of epithelial cadherin (E-cadherin)-mediated cell-cell adhesion or α-catenin depletion prevented CAP350 localisation at cell-cell junctions. Knocking down junction-located CAP350 inhibited the establishment of an apico-basal array of microtubules and impaired the acquisition of columnar shape in Madin-Darby canine kidney II (MDCKII) cells grown as polarised epithelia. Furthermore, MDCKII cystogenesis was also defective in junctional CAP350-depleted cells. CAP350-depleted MDCKII cysts were smaller and contained either multiple lumens or no lumen. Membrane polarity was not affected, but cortical microtubule bundles did not properly form. Our results indicate that CAP350 may act as an adaptor between adherens junctions and microtubules, thus regulating epithelial differentiation and contributing to the definition of cell architecture. We also uncover a central role of α-catenin in global cytoskeleton remodelling, in which it acts not only on actin but also on MT reorganisation during epithelial morphogenesis.


Asunto(s)
Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Microtúbulos/genética , Morfogénesis/genética , Proteínas Nucleares/genética , alfa Catenina/genética , Uniones Adherentes/metabolismo , Uniones Adherentes/ultraestructura , Adipocitos/citología , Adipocitos/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular , Línea Celular , Polaridad Celular , Forma de la Célula , Perros , Embrión no Mamífero , Células Epiteliales/citología , Vectores Genéticos , Humanos , Lentivirus/genética , Células de Riñón Canino Madin Darby , Proteínas de Microtúbulos/antagonistas & inhibidores , Proteínas de Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Oryzias , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Técnicas del Sistema de Dos Híbridos , alfa Catenina/metabolismo
12.
Philos Trans R Soc Lond B Biol Sci ; 369(1650)2014 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-25047616

RESUMEN

A shared feature among all microtubule (MT)-dependent processes is the requirement for MTs to be organized in arrays of defined geometry. At a fundamental level, this is achieved by precisely controlling the timing and localization of the nucleation events that give rise to new MTs. To this end, MT nucleation is restricted to specific subcellular sites called MT-organizing centres. The primary MT-organizing centre in proliferating animal cells is the centrosome. However, the discovery of MT nucleation capacity of the Golgi apparatus (GA) has substantially changed our understanding of MT network organization in interphase cells. Interestingly, MT nucleation at the Golgi apparently relies on multiprotein complexes, similar to those present at the centrosome, that assemble at the cis-face of the organelle. In this process, AKAP450 plays a central role, acting as a scaffold to recruit other centrosomal proteins important for MT generation. MT arrays derived from either the centrosome or the GA differ in their geometry, probably reflecting their different, yet complementary, functions. Here, I review our current understanding of the molecular mechanisms involved in MT nucleation at the GA and how Golgi- and centrosome-based MT arrays work in concert to ensure the formation of a pericentrosomal polarized continuous Golgi ribbon structure, a critical feature for cell polarity in mammalian cells. In addition, I comment on the important role of the Golgi-nucleated MTs in organizing specialized MT arrays that serve specific functions in terminally differentiated cells.


Asunto(s)
Centrosoma/fisiología , Aparato de Golgi/fisiología , Microtúbulos/fisiología , Mitosis/fisiología , Modelos Biológicos , Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas del Citoesqueleto/metabolismo
13.
J Clin Microbiol ; 52(3): 803-13, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24353006

RESUMEN

Recent evidence suggests that most influenza A virus gene segments can contribute to the pathogenicity of the virus. In this regard, the hemagglutinin (HA) subtype of the circulating strains has been closely surveyed, but the reassortment of internal gene segments is usually not monitored as a potential source of an increased pathogenicity. In this work, an oligonucleotide DNA microarray (PhyloFlu) designed to determine the phylogenetic origins of the eight segments of the influenza virus genome was constructed and validated. Clades were defined for each segment and also for the 16 HA and 9 neuraminidase (NA) subtypes. Viral genetic material was amplified by reverse transcription-PCR (RT-PCR) with primers specific to the conserved 5' and 3' ends of the influenza A virus genes, followed by PCR amplification with random primers and Cy3 labeling. The microarray unambiguously determined the clades for all eight influenza virus genes in 74% (28/38) of the samples. The microarray was validated with reference strains from different animal origins, as well as from human, swine, and avian viruses from field or clinical samples. In most cases, the phylogenetic clade of each segment defined its animal host of origin. The genomic fingerprint deduced by the combined information of the individual clades allowed for the determination of the time and place that strains with the same genomic pattern were previously reported. PhyloFlu is useful for characterizing and surveying the genetic diversity and variation of animal viruses circulating in different environmental niches and for obtaining a more detailed surveillance and follow up of reassortant events that can potentially modify virus pathogenicity.


Asunto(s)
Dermatoglifia del ADN/métodos , Genoma Viral , Técnicas de Genotipaje/métodos , Virus de la Influenza A/clasificación , Gripe Humana/virología , Análisis por Micromatrices/métodos , Infecciones por Paramyxoviridae/veterinaria , Animales , Análisis por Conglomerados , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Infecciones por Paramyxoviridae/virología , Filogenia , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virología/métodos
14.
Dev Cell ; 23(4): 782-95, 2012 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-23041384

RESUMEN

Polarized trafficking of adhesion receptors plays a pivotal role in controlling cellular behavior during morphogenesis. Particularly, clathrin-dependent endocytosis of integrins has long been acknowledged as essential for cell migration. However, little is known about the contribution of integrin trafficking to epithelial tissue morphogenesis. Here we show how the transmembrane protein Opo, previously described for its essential role during optic cup folding, plays a fundamental role in this process. Through interaction with the PTB domain of the clathrin adaptors Numb and Numbl via an integrin-like NPxF motif, Opo antagonizes Numb/Numbl function and acts as a negative regulator of integrin endocytosis in vivo. Accordingly, numb/numbl gain-of-function experiments in teleost embryos mimic the retinal malformations observed in opo mutants. We propose that developmental regulator Opo enables polarized integrin localization by modulating Numb/Numbl, thus directing the basal constriction that shapes the vertebrate retina epithelium.


Asunto(s)
Endocitosis , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Epitelio Pigmentado de la Retina/embriología , Epitelio Pigmentado de la Retina/metabolismo , Animales , Células Cultivadas , Proteínas de Peces/antagonistas & inhibidores , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Células HeLa , Humanos , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Oryzias , Epitelio Pigmentado de la Retina/citología
15.
J Neurochem ; 123(4): 635-44, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22913583

RESUMEN

Regulation of proteasome abundance to meet cell needs under stress conditions is critical for maintaining cellular homeostasis. However, the effects of aging on this homeostatic response remain unknown. In this report, we analyzed in young and aged rat hippocampus, the dynamics of proteasome recovery induced by proteasome stress. Proteasome inhibition in young rats leads to an early and coordinate transcriptional and translational up-regulation of both the catalytic subunits of constitutive proteasome and the proteasome maturation protein. By contrast, aged rats up-regulated the inducible catalytic subunits and showed a lower and shorter expression of proteasome maturation protein. This resulted in a faster recovery of proteasome activity in young rats. Importantly, proteasome inhibition highly affected pyramidal cells, leading to the accumulation of ubiquitinated proteins in perinuclear regions of aged, but not young pyramidal neurons. These data strongly suggest that age-dependent differences in proteasome level and composition could contribute to neurodegeneration induced by proteasome dysfunction in normal and pathological aging.


Asunto(s)
Envejecimiento , Hipocampo/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Regulación hacia Arriba/fisiología , Factores de Edad , Animales , Dominio Catalítico/fisiología , Nucléolo Celular/metabolismo , Hipocampo/citología , Inmunoproteínas/metabolismo , Masculino , Neuronas/citología , Neuronas/metabolismo , Ratas , Ratas Wistar
16.
J Neuroinflammation ; 9: 87, 2012 May 04.
Artículo en Inglés | MEDLINE | ID: mdl-22559833

RESUMEN

BACKGROUND: Neuroinflammation and protein accumulation are characteristic hallmarks of both normal aging and age-related neurodegenerative diseases. However, the relationship between these factors in neurodegenerative processes is poorly understood. We have previously shown that proteasome inhibition produced higher neurodegeneration in aged than in young rats, suggesting that other additional age-related events could be involved in neurodegeneration. We evaluated the role of lipopolysaccharide (LPS)-induced neuroinflammation as a potential synergic risk factor for hippocampal neurodegeneration induced by proteasome inhibition. METHODS: Young male Wistar rats were injected with 1 µL of saline or LPS (5 mg/mL) into the hippocampus to evaluate the effect of LPS-induced neuroinflammation on protein homeostasis. The synergic effect of LPS and proteasome inhibition was analyzed in young rats that first received 1 µL of LPS and 24 h later 1 µL (5 mg/mL) of the proteasome inhibitor lactacystin. Animals were sacrificed at different times post-injection and hippocampi isolated and processed for gene expression analysis by real-time polymerase chain reaction; protein expression analysis by western blots; proteasome activity by fluorescence spectroscopy; immunofluorescence analysis by confocal microscopy; and degeneration assay by Fluoro-Jade B staining. RESULTS: LPS injection produced the accumulation of ubiquitinated proteins in hippocampal neurons, increased expression of the E2 ubiquitin-conjugating enzyme UB2L6, decreased proteasome activity and increased immunoproteasome content. However, LPS injection was not sufficient to produce neurodegeneration. The combination of neuroinflammation and proteasome inhibition leads to higher neuronal accumulation of ubiquitinated proteins, predominant expression of pro-apoptotic markers and increased neurodegeneration, when compared with LPS or lactacystin (LT) injection alone. CONCLUSIONS: Our results identify neuroinflammation as a risk factor that increases susceptibility to neurodegeneration induced by proteasome inhibition. These results highlight the modulation of neuroinflammation as a mechanism for neuronal protection that could be relevant in situations where both factors are present, such as aging and neurodegenerative diseases.


Asunto(s)
Hipocampo/efectos de los fármacos , Lipopolisacáridos/toxicidad , Degeneración Nerviosa/inducido químicamente , Inhibidores de Proteasoma/toxicidad , Acetilcisteína/análogos & derivados , Acetilcisteína/toxicidad , Envejecimiento/efectos de los fármacos , Envejecimiento/fisiología , Animales , Sinergismo Farmacológico , Hipocampo/enzimología , Hipocampo/patología , Inflamación/inducido químicamente , Inflamación/epidemiología , Inflamación/patología , Masculino , Degeneración Nerviosa/epidemiología , Degeneración Nerviosa/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Ratas Wistar , Ubiquitinación/efectos de los fármacos , Ubiquitinación/fisiología
17.
Neurobiol Aging ; 33(10): 2462-77, 2012 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-22177721

RESUMEN

Lewy bodies containing the centrosomal protein γ-tubulin and fragmentation of Golgi apparatus (GA) have been described in nigral neurons of Parkinson's disease (PD) patients. However, the relevance of these features in PD pathophysiology remains unknown. We analyzed the impact of proteasome inhibition in the formation of γ-tubulin-containing aggregates as well as on GA structure. SH-SY5Y cells were treated with the proteasome inhibitor Z-Leu-Leu-Leu-al (MG132) to induce centrosomal-protein aggregates. Then, microtubules (MTs) and Golgi dynamics, as well as the vesicular transport of dopamine transporter (DAT) were evaluated both in vitro and in living cells. MG132 treatment induced γ-tubulin aggregates which altered microtubule nucleation. MG132-treated cells containing γ-tubulin aggregates showed fragmentation of GA and perturbation of the trans-Golgi network. Under these conditions, the DAT accumulated at the centrosomal-Golgi region indicating that the vesicular transport of DAT was disrupted. Thus, centrosomal aggregates and fragmentation of GA are 2 closely related processes that could result in the disruption of the vesicular transport of DAT toward the plasma membrane in a model of dopaminergic neuronal degeneration.


Asunto(s)
Centrosoma/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Aparato de Golgi/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Línea Celular , Centrosoma/efectos de los fármacos , Aparato de Golgi/efectos de los fármacos , Humanos , Leupeptinas/farmacología , Microtúbulos/metabolismo , Inhibidores de Proteasoma/farmacología , Tubulina (Proteína)/metabolismo
18.
Mol Biol Cell ; 22(22): 4302-11, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21937724

RESUMEN

Pituitary tumor transforming gene 1 (PTTG1), also known as securin, has been implicated in many biological functions, including inhibition of sister chromatid separation, DNA repair, organ development, and regulation of the expression and secretion of angiogenic and metastatic factors. Although most of these functions of securin seem to depend on the localization of PTTG1 in the nucleus of the cell, a fraction of the protein has been also detected in the cytoplasm. Here we demonstrate that, in different cell types, a portion of cytoplasmic PTTG1 is associated with the cis face of the Golgi apparatus and that this localization depends on PTTG1 phosphorylation status. In this organelle, PTTG1 forms a complex with proteins involved in microtubule nucleation, including GM130, AKAP450, and γ-tubulin. RNA interference-mediated depletion of PTTG1 produces a delay in centrosomal and noncentrosomal microtubule nucleation. Cells lacking PTTG1 show severe defects in both cell polarization and migration in wound-healing assays. To our knowledge, this is the first study reporting the role of PTTG1 in microtubule nucleation and cell polarization, two processes directly involved in cell migration. We believe that these findings will contribute to understanding the mechanisms underlying PTTG1-mediated biological functions.


Asunto(s)
Movimiento Celular , Aparato de Golgi/metabolismo , Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Anclaje a la Quinasa A/metabolismo , Anticuerpos Monoclonales/inmunología , Autoantígenos/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Polaridad Celular , Centrosoma , Proteínas del Citoesqueleto/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño , Securina , Tubulina (Proteína)/metabolismo , Cicatrización de Heridas
19.
J Cell Biol ; 193(5): 917-33, 2011 May 30.
Artículo en Inglés | MEDLINE | ID: mdl-21606206

RESUMEN

Mammalian cells exhibit a frequent pericentrosomal Golgi ribbon organization. In this paper, we show that two AKAP450 N-terminal fragments, both containing the Golgi-binding GM130-interacting domain of AKAP450, dissociated endogenous AKAP450 from the Golgi and inhibited microtubule (MT) nucleation at the Golgi without interfering with centrosomal activity. These two fragments had, however, strikingly different effects on both Golgi apparatus (GA) integrity and positioning, whereas the short fragment induced GA circularization and ribbon fragmentation, the large construct that encompasses an additional p150glued/MT-binding domain induced separation of the Golgi ribbon from the centrosome. These distinct phenotypes arose by specific interference of each fragment with either Golgi-dependent or centrosome-dependent stages of Golgi assembly. We could thus demonstrate that breaking the polarity axis by perturbing GA positioning has a more dramatic effect on directional cell migration than disrupting the Golgi ribbon. Both features, however, were required for ciliogenesis. We thus identified AKAP450 as a key determinant of pericentrosomal Golgi ribbon integrity, positioning, and function in mammalian cells.


Asunto(s)
Movimiento Celular , Centrosoma/metabolismo , Cilios/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Anclaje a la Quinasa A/metabolismo , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Humanos , Microtúbulos/metabolismo
20.
Dev Dyn ; 240(3): 723-36, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21337470

RESUMEN

We describe the localization of the golgin GMAP210 and the intraflagellar protein IFT88 in the Golgi of spermatids and the participation of these two proteins in the development of the acrosome-acroplaxome complex, the head-tail coupling apparatus (HTCA) and the spermatid tail. Immunocytochemical experiments show that GMAP210 predominates in the cis-Golgi, whereas IFT88 prevails in the trans-Golgi network. Both proteins colocalize in proacrosomal vesicles, along acrosome membranes, the HTCA and the developing tail. IFT88 persists in the acrosome-acroplaxome region of the sperm head, whereas GMAP210 is no longer seen there. Spermatids of the Ift88 mouse mutant display abnormal head shaping and are tail-less. GMAP210 is visualized in the Ift88 mutant during acrosome-acroplaxome biogenesis. However, GMAP210-stained vesicles, mitochondria and outer dense fiber material build up in the manchette region and fail to reach the abortive tail stump in the mutant. In vitro disruption of the spermatid Golgi and microtubules with Brefeldin-A and nocodazole blocks the progression of GMAP210- and IFT88-stained proacrosomal vesicles to the acrosome-acroplaxome complex but F-actin distribution in the acroplaxome is not affected. We provide the first evidence that IFT88 is present in the Golgi of spermatids, that the microtubule-associated golgin GMAP210 and IFT88 participate in acrosome, HTCA, and tail biogenesis, and that defective intramanchette transport of cargos disrupts spermatid tail development.


Asunto(s)
Acrosoma/metabolismo , Aparato de Golgi/metabolismo , Proteínas Nucleares/metabolismo , Espermátides/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Acrosoma/ultraestructura , Actinas/metabolismo , Animales , Brefeldino A/farmacología , Proteínas del Citoesqueleto , Técnica del Anticuerpo Fluorescente Indirecta , Aparato de Golgi/ultraestructura , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Microscopía Electrónica , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Nocodazol/farmacología , Proteínas Nucleares/genética , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espermátides/ultraestructura , Proteínas Supresoras de Tumor/genética
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