RESUMEN
The yeast Sup35 protein misfolds into the infectious [PSI+] prion, which is then propagated by the severing activity of the molecular chaperone, Hsp104. Unlike other yeast prions, this prion is unique in that it is efficiently cured by the overexpression as well as the inactivation of Hsp104. However, it is controversial whether curing by overexpression is due to the dissolution of the prion seeds by the trimming activity of Hsp104 or the asymmetric segregation of the prion seeds between mother and daughter cells which requires cell division. To answer this question, we conducted experiments and found no difference in the extent of curing between mother and daughter cells when half of the cells were cured by Hsp104 overexpression in one generation. Furthermore, curing was not affected by the lack of Sir2 expression, which was reported to be required for asymmetric segregation of the [PSI+] seeds. More importantly, when either hydroxyurea or ethanol were used to inhibit cell division, the extent of curing by Hsp104 overexpression was not significantly reduced. Therefore, the curing of [PSI+] by Hsp104 overexpression is not due to asymmetric segregation of the prion seeds, but rather their dissolution by Hsp104.
Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Priones/metabolismo , SolubilidadRESUMEN
OBJECTIVE: Neonatal lupus erythematosus (NLE) may develop after transplacental transfer of maternal autoantibodies with cardiac manifestations (congenital heart block, CHB) including atrioventricular block, atrial and ventricular arrhythmias, and cardiomyopathies. The association with anti-Ro/SSA antibodies is well established, but a recurrence rate of only 12%-16% despite persisting maternal autoantibodies suggests that additional factors are required for CHB development. Here, we identify fetal genetic variants conferring risk of CHB and elucidate their effects on cardiac function. METHODS: A genome-wide association study was performed in families with at least one case of CHB. Gene expression was analysed by microarrays, RNA sequencing and PCR and protein expression by western blot, immunohistochemistry, immunofluorescence and flow cytometry. Calcium regulation and connectivity were analysed in primary cardiomyocytes and cells induced from pleuripotent stem cells. Fetal heart performance was analysed by Doppler/echocardiography. RESULTS: We identified DNAJC6 as a novel fetal susceptibility gene, with decreased cardiac expression of DNAJC6 associated with the disease risk genotype. We further demonstrate that fetal cardiomyocytes deficient in auxilin, the protein encoded by DNAJC6, have abnormal connectivity and Ca2+ homoeostasis in culture, as well as decreased cell surface expression of the Cav1.3 calcium channel. Doppler echocardiography of auxilin-deficient fetal mice revealed cardiac NLE abnormalities in utero, including abnormal heart rhythm with atrial and ventricular ectopias, as well as a prolonged atrioventricular time intervals. CONCLUSIONS: Our study identifies auxilin as the first genetic susceptibility factor in NLE modulating cardiac function, opening new avenues for the development of screening and therapeutic strategies in CHB.
Asunto(s)
Bloqueo Atrioventricular , Auxilinas , Animales , Anticuerpos Antinucleares , Bloqueo Atrioventricular/genética , Autoanticuerpos , Corazón Fetal , Estudio de Asociación del Genoma Completo , Bloqueo Cardíaco/congénito , Lupus Eritematoso Sistémico/congénito , RatonesRESUMEN
Prions are infectious proteins that self-propagate by changing from their normal folded conformation to a misfolded conformation. The misfolded conformation, which is typically rich in ß-sheet, serves as a template to convert the prion protein into its misfolded conformation. In yeast, the misfolded prion proteins are assembled into amyloid fibers or seeds, which are constantly severed and transmitted to daughter cells. To cure prions in yeast, it is necessary to eliminate all the prion seeds. Multiple mechanisms of curing have been found including inhibiting severing of the prion seeds, gradual dissolution of the prion seeds, asymmetric segregation of the prion seeds between mother and daughter cells during cell division, and degradation of the prion seeds. These mechanisms, achieved by using different protein quality control machinery, are not mutually exclusive; depending on conditions, multiple mechanisms may work simultaneously to achieve curing. This review discusses the various methods that have been used to differentiate between these mechanisms of curing.
Asunto(s)
Priones/genética , Priones/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Choque Térmico/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/patogenicidad , Proteolisis , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The [URE3] yeast prion is the self-propagating amyloid form of the Ure2 protein. [URE3] is cured by overexpression of several yeast proteins, including Ydj1, Btn2, Cur1, Hsp42, and human DnaJB6. To better understand [URE3] curing, we used real-time imaging with a yeast strain expressing a GFP-labeled full-length Ure2 construct to monitor the curing of [URE3] over time. [URE3] yeast cells exhibited numerous fluorescent foci, and expression of the GFP-labeled Ure2 affected neither mitotic stability of [URE3] nor the rate of [URE3] curing by the curing proteins. Using guanidine to cure [URE3] via Hsp104 inactivation, we found that the fluorescent foci are progressively lost as the cells divide until they are cured; the fraction of cells that retained the foci was equivalent to the [URE3] cell fraction measured by a plating assay, indicating that the foci were the prion seeds. During the curing of [URE3] by Btn2, Cur1, Hsp42, or Ydj1 overexpression, the foci formed aggregates, many of which were 0.5 µm or greater in size, and [URE3] was cured by asymmetric segregation of the aggregated seeds. In contrast, DnaJB6 overexpression first caused a loss of detectable foci in cells that were still [URE3] before there was complete dissolution of the seeds, and the cells were cured. We conclude that GFP labeling of full-length Ure2 enables differentiation among the different [URE3]-curing mechanisms, including inhibition of severing followed by seed dilution, seed clumping followed by asymmetric segregation between mother and daughter cells, and seed dissolution.
Asunto(s)
Proteínas Fúngicas/metabolismo , Imagen Molecular , Priones/metabolismo , Levaduras/citología , Factores de Tiempo , Levaduras/metabolismoRESUMEN
Prions arise from proteins that have two possible conformations: properly folded and non-infectious or misfolded and infectious. The [PSI+] yeast prion, which is the misfolded and self-propagating form of the translation termination factor eRF3 (Sup35), can be cured of its infectious conformation by overexpression of Hsp104, which helps dissolve the prion seeds. This dissolution depends on the trimming activity of Hsp104, which reduces the size of the prion seeds without increasing their number. To further understand the relationship between trimming and curing, trimming was followed by measuring the loss of GFP-labeled Sup35 foci from both strong and weak [PSI+] variants; the former variant has more seeds and less soluble Sup35 than the latter. Overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) trimmed the weak [PSI+] variants much faster than the strong variants and cured the weak variants an order of magnitude faster than the strong variants. Overexpression of the fungal Hsp104 homologs from Schizosaccharomyces pombe (Sp-Hsp104) or Candida albicans (Ca-Hsp104) also trimmed and cured the weak variants, but interestingly, it neither trimmed nor cured the strong variants. These results show that, because Sc-Hsp104 has greater trimming activity than either Ca-Hsp104 or Sp-Hsp104, it cures both the weak and strong variants, whereas Ca-Hsp104 and Sp-Hsp104 only cure the weak variants. Therefore, curing by Hsp104 overexpression depends on both the trimming ability of the fungal Hsp104 homolog and the strength of the [PSI+] variant: the greater the trimming activity of the Hsp104 homolog and the weaker the variant, the greater the curing.
Asunto(s)
Proteínas de Choque Térmico/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Prueba de Complementación Genética , Proteínas de Choque Térmico/genética , Factores de Terminación de Péptidos/genética , Priones/genética , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
The conversion of the properly folded prion protein, PrPc, to its misfolded amyloid form, PrPsc, occurs as the two proteins traffic along the endocytic pathway and PrPc is exposed to PrPsc. To determine the specific site of prion conversion, we knocked down various proteins in the endocytic pathway including Rab7a, Tsg101 and Hrs (also known as HGS). PrPsc was markedly reduced in two chronically infected cell lines by preventing the maturation of the multivesicular body, a process that begins in the early endosome and ends with the sorting of cargo to the lysosome. By contrast, knocking down proteins in the retromer complex, which diverts cargo away from the multivesicular body caused an increase in PrPsc levels. These results suggest that the multivesicular body is the major site for intracellular conversion of PrPc to PrPsc.
Asunto(s)
Cuerpos Multivesiculares/metabolismo , Priones/metabolismo , Animales , Encéfalo/metabolismo , Lisosomas/metabolismo , Ratones , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Priones/genética , Procesamiento Proteico-PostraduccionalRESUMEN
Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.
Asunto(s)
Encéfalo/metabolismo , Clatrina/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Auxilinas/genética , Auxilinas/metabolismo , Clatrina/genética , Proteínas del Choque Térmico HSC70/genética , Ratones , Ratones Noqueados , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Transporte de Proteínas/fisiologíaRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson disease (PD), and common variants around LRRK2 are a risk factor for sporadic PD. Using protein-protein interaction arrays, we identified BCL2-associated athanogene 5, Rab7L1 (RAB7, member RAS oncogene family-like 1), and Cyclin-G-associated kinase as binding partners of LRRK2. The latter two genes are candidate genes for risk for sporadic PD identified by genome-wide association studies. These proteins form a complex that promotes clearance of Golgi-derived vesicles through the autophagy-lysosome system both in vitro and in vivo. We propose that three different genes for PD have a common biological function. More generally, data integration from multiple unbiased screens can provide insight into human disease mechanisms.
Asunto(s)
Sitios Genéticos/genética , Predisposición Genética a la Enfermedad/genética , Complejos Multiproteicos/metabolismo , Enfermedad de Parkinson/enzimología , Mapeo de Interacción de Proteínas/métodos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Análisis de Varianza , Western Blotting , Encéfalo/metabolismo , Fraccionamiento Celular , Cartilla de ADN/genética , Estudio de Asociación del Genoma Completo/métodos , Aparato de Golgi/ultraestructura , Células HEK293 , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Espectrometría de Masas , Microscopía Confocal , Complejos Multiproteicos/genética , Plásmidos/genética , Proteínas Serina-Treonina Quinasas/genética , Vesículas Transportadoras/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
This article conveys the authors' perspectives and insights gained during the development and implementation of their experience with a community-based intervention designed to address obesity among Black women. The reader gets a look at the authors' personal weight struggles, as the intervention they designed to help others transformed their lives as well as the participants. The authors also shared their perspectives on the impact of religion and spirituality on healthy lifestyles.
Asunto(s)
Negro o Afroamericano/psicología , Características Culturales , Hispánicos o Latinos/psicología , Obesidad/etnología , Obesidad/psicología , Espiritualidad , Población Blanca/psicología , Adulto , Negro o Afroamericano/estadística & datos numéricos , Anciano , Anciano de 80 o más Años , Femenino , Hispánicos o Latinos/estadística & datos numéricos , Humanos , Persona de Mediana Edad , Estados Unidos , Población Blanca/estadística & datos numéricosRESUMEN
The Hsc70 cochaperone, G cyclin-associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin-dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin-mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin-dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.
Asunto(s)
Senescencia Celular , Centrosoma/metabolismo , Clatrina/metabolismo , Endocitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Complejo 2 de Proteína Adaptadora/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Línea Celular , Clatrina/genética , Daño del ADN , Lisosomas/metabolismo , Ratones , Fenotipo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Transporte de Proteínas , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The [PSI(+)] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [PSI(+)], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [PSI(+)]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [PSI(+)] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [PSI(+)] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.
Asunto(s)
Proteínas de Choque Térmico/genética , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Expresión Génica , Proteínas de Choque Térmico/metabolismo , Factores de Terminación de Péptidos/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , SolubilidadRESUMEN
Clathrin coats, which stabilize membrane curvature during endocytosis and vesicular trafficking, form highly polymorphic fullerene lattices. We used cryo-electron tomography to visualize coated particles in isolates from bovine brain. The particles range from â¼66 to â¼134nm in diameter, and only 20% of them (all ⩾80nm) contain vesicles. The remaining 80% are clathrin "baskets", presumably artifactual assembly products. Polyhedral models were built for 54 distinct coat geometries. In true coated vesicles (CVs), most vesicles are offset to one side, leaving a crescent of interstitial space between the coat and the membrane for adaptor proteins and other components. The latter densities are fewer on the membrane-proximal side, which may represent the last part of the vesicle to bud off. A small number of densities - presumably cargo proteins - are associated with the interior surface of the vesicles. The clathrin coat, adaptor proteins, and vesicle membrane contribute almost all of the mass of a CV, with most cargoes accounting for only a few percent. The assembly of a CV therefore represents a massive biosynthetic effort to internalize a relatively diminutive payload. Such a high investment may be needed to overcome the resistance of membranes to high curvature.
Asunto(s)
Vesículas Cubiertas por Clatrina/metabolismo , Animales , Encéfalo/metabolismo , Bovinos , Tomografía con Microscopio Electrónico/métodos , ElectronesRESUMEN
Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Fragmentos de Péptidos/metabolismo , Factores de Terminación de Péptidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Western Blotting , Glutamina/genética , Humanos , Proteína Huntingtina , Microscopía Confocal , Mutación , Proteínas del Tejido Nervioso/genética , Fragmentos de Péptidos/genética , Factores de Terminación de Péptidos/genética , Plásmidos/genética , Priones/genética , Priones/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Transformación Genética , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
Neuronally expressed auxilin and ubiquitously expressed cyclin-G-dependent kinase (GAK) are homologous proteins that act as cochaperones to support the Hsc70-dependent clathrin uncoating of clathrin-coated vesicles. GAK was previously shown to be essential in mouse during embryonic development and in the adult. We have now engineered an auxilin knockout mouse. Mutant mice had a high rate of early postnatal mortality and surviving pups generally had a lower body weight than wild-type pups, although they had a normal life span. GAK was up-regulated as much as 3-fold in the brains of both surviving neonates and adult mutant mice. An increased number of clathrin-coated vesicles and empty cages were present at knockout synapses both in situ and in primary neuronal cultures. Additionally, clathrin-mediated endocytosis of synaptic vesicles in knockout hippocampal neurons was impaired, most likely due to sequestration of coat components in assembled coats and cages. Collectively, our results demonstrate the specialized role of auxilin in the recycling of synaptic vesicles at synapses, but also show that its function can be partially compensated for by up-regulation of GAK.
Asunto(s)
Auxilinas/fisiología , Clatrina/metabolismo , Endocitosis , Sinapsis/metabolismo , Animales , Auxilinas/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Terminaciones Nerviosas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Regulación hacia ArribaRESUMEN
Hsp104 propagates the yeast prion [PSI+], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [PSI+] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation. Investigating this question, we now find, first, that mutating this site prevents both the curing of [PSI+] by Hsp104 overexpression and the trimming activity of Hsp104. Second, we find that depending on the specific member of the Hsp70 family binding to the N-terminal domain of Hsp104, both trimming and the curing caused by Hsp104 overexpression are either increased or decreased in parallel. Therefore, the binding of Hsp70 to the N-terminal domain of Hsp104 regulates both the rate of [PSI+] trimming by Hsp104 and the rate of [PSI+] curing by Hsp104 overexpression.
Asunto(s)
Priones , Proteínas de Saccharomyces cerevisiae , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Priones/genética , Priones/metabolismo , Factores de Terminación de Péptidos/químicaRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder characterized by defective dopaminergic (DAergic) input to the striatum. Mutations in two genes encoding synaptically enriched clathrin-uncoating factors, synaptojanin 1 (SJ1) and auxilin, have been implicated in atypical Parkinsonism. SJ1 knock-in (SJ1-KIRQ) mice carrying a disease-linked mutation display neurological manifestations reminiscent of Parkinsonism. Here we report that auxilin knockout (Aux-KO) mice display dystrophic changes of a subset of nigrostriatal DAergic terminals similar to those of SJ1-KIRQ mice. Furthermore, Aux-KO/SJ1-KIRQ double mutant mice have shorter lifespan and more severe synaptic defects than single mutant mice. These include increase in dystrophic striatal nerve terminals positive for DAergic markers and for the PD risk protein SV2C, as well as adaptive changes in striatal interneurons. The synergistic effect of the two mutations demonstrates a special lability of DAergic neurons to defects in clathrin uncoating, with implications for PD pathogenesis in at least some forms of this condition.
RESUMEN
The Sup35 prion protein of budding yeast has been reported to undergo phase separation to form liquid droplets both at low pH in vitro and when energy depletion decreases the intracellular pH in vivo. It also has been shown using purified proteins that this phase separation is driven by the prion domain of Sup35 and does not re-quire its C-terminal domain. In contrast, we now find that a Sup35 fragment consisting of only the N-terminal prion domain and the M-domain does not phase separate in vivo; this phase separation of Sup35 requires the C-terminal domain, which binds Sup45 to form the translation termination complex. The phase-separated Sup35 not only colocalizes with Sup45 but also with Pub1, a stress granule marker protein. In addition, like stress granules, phase separation of Sup35 appears to require mRNA since cycloheximide treatment, which inhibits mRNA release from ribosomes, prevents phase separation of Sup35. Finally, unlike Sup35 in vitro, Sup35 condensates do not disassemble in vivo when the intracellular pH is increased. These results suggest that, in energy-depleted cells, Sup35 forms supramolecular assemblies that differ from the Sup35 liquid droplets that form in vitro.
RESUMEN
Auxilin participates in the uncoating of clathrin-coated vesicles (CCVs), thereby facilitating synaptic vesicle (SV) regeneration at presynaptic sites. Auxilin (DNAJC6/PARK19) loss-of-function mutations cause early-onset Parkinson's disease (PD). Here, we utilized auxilin knockout (KO) mice to elucidate the mechanisms through which auxilin deficiency and clathrin-uncoating deficits lead to PD. Auxilin KO mice display cardinal features of PD, including progressive motor deficits, α-synuclein pathology, nigral dopaminergic loss, and neuroinflammation. Significantly, treatment with L-DOPA ameliorated motor deficits. Unbiased proteomic and neurochemical analyses of auxilin KO brains indicated dopamine dyshomeostasis. We validated these findings by demonstrating slower dopamine reuptake kinetics in vivo, an effect associated with dopamine transporter misrouting into axonal membrane deformities in the dorsal striatum. Defective SV protein sorting and elevated synaptic autophagy also contribute to ineffective dopamine sequestration and compartmentalization, ultimately leading to neurodegeneration. This study provides insights into how presynaptic endocytosis deficits lead to dopaminergic vulnerability and pathogenesis of PD.
Asunto(s)
Enfermedad de Parkinson , Ratones , Animales , Enfermedad de Parkinson/patología , Vesículas Sinápticas/metabolismo , Auxilinas/genética , Auxilinas/metabolismo , Dopamina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Proteómica , Transporte de Proteínas , Sustancia Negra/metabolismoRESUMEN
During mitosis, all chromosomes must attach to microtubules of the mitotic spindle to ensure correct chromosome segregation. Microtubule attachment occurs at specialized structures at the centromeric region of chromosomes, called kinetochores. These kinetochores can generate microtubule attachments through capture of centrosome-derived microtubules, but in addition, they can generate microtubules themselves, which are subsequently integrated with centrosome-derived microtubules to form the mitotic spindle. Here, we have performed a large scale RNAi screen and identify cyclin G-associated kinase (GAK) as a novel regulator of microtubule generation at kinetochores/chromatin. This function of GAK requires its C-terminal J-domain, which is essential for clathrin recycling from endocytic vesicles. Consistently, cells lacking GAK show strongly reduced levels of clathrin on the mitotic spindle, and reduction of clathrin levels also inhibits microtubule generation at kinetochores/chromosomes. Finally, we present evidence that association of clathrin with the spindle is promoted by a signal coming from the chromosomes. These results identify a role for GAK and clathrin in microtubule outgrowth from kinetochores/chromosomes and suggest that GAK acts through clathrin to control microtubule outgrowth around chromosomes.
Asunto(s)
Clatrina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Huso Acromático/metabolismo , Línea Celular Tumoral , Centrómero/metabolismo , Segregación Cromosómica/fisiología , Cromosomas Humanos/genética , Cromosomas Humanos/fisiología , Cromosomas Humanos/ultraestructura , Clatrina/genética , Clatrina/fisiología , Células HeLa , Humanos , Cinetocoros/fisiología , Microtúbulos/ultraestructura , Mitosis , ARN Interferente Pequeño , Huso Acromático/genética , Tubulina (Proteína)/metabolismoRESUMEN
To understand the role of clathrin-mediated endocytosis in the internalization of normal cellular prion protein (PrP(c)) in neuronal cells, N2a cells were depleted of clathrin by RNA interference. PrP(c) internalization via the constitutive endocytic pathway in the absence of Cu(2+) and the stimulated pathway in the presence of Cu(2+) were measured in both control and clathrin-depleted cells. Depletion of clathrin had almost no effect on the internalization of PrP(c) either in the presence or absence of Cu(2+), in contrast to the marked reduction observed in transferrin uptake. By contrast, the internalization of PrP(c) was inhibited by the raft-disrupting drugs filipin and nystatin, and by the dominant-negative dynamin-1 mutant dynamin-1 K44A, both in the presence and absence of Cu(2+). The internalized PrP(c) was found to colocalize with cargo that traffic in the Arf6 pathway and in large vacuoles in cells expressing the Arf6 dominant-active mutant. These results show that PrP(c) is internalized in a clathrin-independent pathway that is associated with Arf6.