RESUMEN
Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X-ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP-A histone chaperone, binds both CENP-A and the centromere receptor CENP-C without the requirement for the Mis18 complex. While an N-terminal CAL1 fragment wraps around CENP-A/H4 through multiple physical contacts, a C-terminal CAL1 fragment directly binds a CENP-C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP-A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP-C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP-C dimer. Overall, by demonstrating how CAL1 binds CENP-A/H4 and CENP-C, we provide key insights into the minimalistic principles underlying centromere maintenance.
Asunto(s)
Proteína A Centromérica/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Centrómero/química , Centrómero/metabolismo , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación , Unión ProteicaRESUMEN
The single-nucleotide polymorphism (SNP) rs12979860 near the IL28B gene has been associated with the spontaneous clearance of hepatitis C virus. We sought to determine whether this SNP could be associated with the spontaneous control of human immunodeficiency virus (HIV) infection. We studied the prevalence of the IL28B CC genotype among 53 white HIV controllers, compared with the prevalence among 389 HIV-infected noncontrollers. We found that the IL28B CC genotype was independently associated with spontaneous HIV control (odds ratio [OR], 2.669; P = .017), as were female sex (OR, 7.077; P ≤ .001) and the presence of HLA-B57 and/or B27 (OR, 3.080; P = .017). This result supports the idea that common host mechanisms are involved in the spontaneous control of these 2 chronic infections.
Asunto(s)
Infecciones por VIH/genética , Infecciones por VIH/inmunología , Interleucinas/genética , Polimorfismo de Nucleótido Simple/genética , Población Blanca/genética , Adulto , Femenino , Genotipo , Infecciones por VIH/virología , Antígenos HLA-B/genética , Hepatitis C Crónica/genética , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Humanos , Interferones , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
In cultured cerebellar granule neurons, low neuronal activity triggers the intrinsic program of apoptosis, which requires protein synthesis-dependent BAX translocation to mitochondria, a process that may underlie neuronal damage in neurodegeneration. However, the mechanisms that link neuronal activity with the induction of the mitochondrial program of apoptosis remain unclear. Neuronal pentraxin 1 (NP1) is a pro-apoptotic protein induced by low neuronal activity that is increased in damaged neurites in Alzheimer's disease-affected brains. Here we report that NP1 facilitates the accumulation of BAX in mitochondria and regulates mitochondrial dynamics during apoptosis in rat and mouse cerebellar granule neurons in culture. Reduction of neuronal activity increases NP1 protein levels in mitochondria and contributes to mitochondrial fragmentation in a Bax-dependent manner. In addition, NP1 is involved in mitochondrial transport in healthy neurons. These results show that NP1 is targeted to mitochondria acting upstream of BAX and uncover a novel function for NP1 in the regulation of mitochondrial dynamics and trafficking during apoptotic neurodegeneration.
Asunto(s)
Proteína C-Reactiva/fisiología , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Simulación de Dinámica Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
The identification of modulators for proteins without assayable biochemical activity remains a challenge in chemical biology. The presented approach adapts a high-throughput fluorescence binding assay and functional chromatography, two protein-resin technologies, enabling the discovery and isolation of fluorescent natural product probes that target proteins independently of biochemical function. The resulting probes also suggest targetable pockets for lead discovery. Using human survivin as a model, we demonstrate this method with the discovery of members of the prodiginine family as fluorescent probes to the cancer target survivin.
RESUMEN
Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromosomas/metabolismo , Nucleosomas/metabolismo , Animales , Aurora Quinasa B/metabolismo , División Celular , Centrómero/ultraestructura , Segregación Cromosómica , Células HeLa , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Espectrometría de Masas , Microscopía Fluorescente , Mitosis , Fosforilación , Unión Proteica , Pliegue de Proteína , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Survivin/metabolismo , Xenopus laevisRESUMEN
Accumulation of amyloid-beta (Abeta) is thought to play a central role in the progressive loss of synapses, the neurite damage, and the neuronal death that are characteristic in brains affected by Alzheimer's disease. However, the mechanisms through which Abeta produces such neurotoxicity remain unclear. Because Abeta depresses synaptic activity, we investigated whether the neurotoxicity of Abeta depends on the expression of NP1, a protein involved in excitatory synapse remodeling that has recently been shown to mediate neuronal death induced by reduction in neuronal activity in mature neurons. We found that treatment of cortical neurons in culture with Abeta produces a marked increase in NP1 protein that precedes apoptotic neurotoxicity. Silencing NP1 gene expression by RNA interference (short hairpin RNA for RNA interference) prevents the loss of synapses, the reduction in neurite outgrowth, and the apoptosis evoked by Abeta. Transgene overexpression of NP1 reproduced these neurotoxic effects of Abeta. Moreover, we found that NP1 was increased in dystrophic neurites of brains from patients with sporadic late-onset Alzheimer's disease. Dual immunohistochemistry for NP1 and tau showed that NP1 colocalizes with tau deposits in dystrophic neurites. Furthermore, NP1 colocalized with SNAP-25 (synaptosomal-associated protein of 25 kDa) in the majority of dystrophic neurites surrounding amyloid deposits. NP1 was also increased in cell processes surrounding amyloid plaques in the cerebral cortex and hippocampus of APP/PS1 (mutant amyloid precursor protein/presenilin 1) transgenic mice. These findings show that NP1 is a key factor for the synapse loss, the neurite damage, and the apoptotic neuronal death evoked by Abeta and indicate that Abeta contributes to the pathology of Alzheimer's disease by regulating NP1 expression.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Encéfalo/metabolismo , Encéfalo/patología , Proteína C-Reactiva/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuritas/metabolismo , Neuritas/patología , Anciano , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/biosíntesis , Animales , Proteína C-Reactiva/biosíntesis , Proteína C-Reactiva/genética , Células Cultivadas , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Sprague-Dawley , Sinapsis/patología , Regulación hacia Arriba/fisiologíaRESUMEN
TD-60 (also known as RCC2) is a highly conserved protein that structurally resembles the Ran guanine exchange factor (GEF) RCC1, but has not previously been shown to have GEF activity. TD-60 has a typical chromosomal passenger complex (CPC) distribution in mitotic cells, but associates with integrin complexes and is involved in cell motility during interphase. Here we show that TD-60 exhibits GEF activity, in vitro and in cells, for the small GTPase RalA. TD-60 or RalA depletion causes spindle abnormalities in prometaphase associated with abnormal centromeric accumulation of CPC components. TD-60 and RalA apparently work together to contribute to the regulation of kinetochore-microtubule interactions in early mitosis. Importantly, several mitotic phenotypes caused by TD-60 depletion are reverted by the expression of a GTP-locked mutant, RalA (Q72L). The demonstration that a small GTPase participates in the regulation of the CPC reveals a level of mitotic regulation not suspected in previous studies.
Asunto(s)
Proteínas Cromosómicas no Histona/genética , Factores de Intercambio de Guanina Nucleótido/genética , Mitosis/genética , Huso Acromático/metabolismo , Proteínas de Unión al GTP ral/genética , Aurora Quinasa B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Proteínas Inhibidoras de la Apoptosis/metabolismo , Prometafase/genética , Survivin , Proteínas de Unión al GTP ral/metabolismoRESUMEN
Hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a key role in regulating the adaptive response to hypoxia. HIF-1α is stabilised during hypoxia and, after dimerisation with hypoxia-inducible factor 1ß (HIF-1ß), triggers the expression of various genes involved in cell cycle control and energy metabolism associated with cell survival. However, HIF-1α also regulates the expression of proapoptotic genes. The aim of this study was to ascertain the influence of HIF-1α on neurotoxicity evoked by hypoxia in rat cortical neurons. We found that mild hypoxia induces time-dependent neuronal death involving free radical production, mitochondrial depolarisation, cytochrome c release and caspase-3 activation. Lentivirus-mediated HIF-1α knockdown markedly strengthened all of these effects during the initial 24h of hypoxia, which suggests that HIF-1α plays a neuroprotective role in hypoxia-mediated neuronal death. After this initial period, the protective actions of HIF-1α disappeared over the course of the hypoxia-mediated HIF-1α stabilisation. Moreover, lentiviral-mediated overexpression of HIF-1α increased lactate dehydrogenase (LDH) A, one of the target genes for HIF-1α, but did not show protective actions on hypoxia-mediated neuronal death, indicating that the level of endogenous HIF-1α stabilisation achieved during hypoxia was already the maximum required for HIF-1α transcription activities. These results indicate that HIF-1α is neuroprotective in the early phases of hypoxia.