RESUMEN
Nutritional influences have been discussed as potential modulators of Parkinson's disease (PD) pathology through various epidemiological and physiological studies. In animal models, a high-fat diet (HFD) with greater intake of lipid-derived calories leads to accelerated disease onset and progression. The underlying molecular mechanisms of HFD-induced aggravated pathology, however, remain largely unclear. In this study, we aimed to further illuminate the effects of a fat-enriched diet in PD by examining the brainstem and hippocampal transcriptome of alpha-synuclein transgenic mice exposed to a life-long HFD. Investigating individual transcript isoforms, differential gene expression and co-expression clusters, we observed that transcriptional differences between wild-type (WT) and transgenic animals intensified in both regions under HFD. Both brainstem and hippocampus displayed strikingly similar transcriptomic perturbation patterns. Interestingly, expression differences resulted mainly from responses in WT animals to HFD, while these genes remained largely unchanged or were even slightly oppositely regulated by diet in transgenic animals. Genes and co-expressed gene groups exhibiting this dysregulation were linked to metabolic and mitochondrial pathways. Our findings propose the failure of metabolic adaptions as the potential explanation for accelerated disease unfolding under exposure to HFD. From the identified clusters of co-expressed genes, several candidates lend themselves to further functional investigations.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Ratones , Animales , Ratones Transgénicos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Dieta Alta en Grasa/efectos adversos , Perfilación de la Expresión Génica , Ratones Endogámicos C57BLRESUMEN
Single-cell transcriptomics has revealed specific glial activation states associated with the pathogenesis of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. While these findings may eventually lead to new therapeutic opportunities, little is known about how these glial responses are reflected by biomarker changes in bodily fluids. Such knowledge, however, appears crucial for patient stratification, as well as monitoring disease progression and treatment responses in clinical trials. Here, we took advantage of well-described mouse models of ß-amyloidosis and α-synucleinopathy to explore cerebrospinal fluid (CSF) proteome changes related to their respective proteopathic lesions. Nontargeted liquid chromatography-mass spectrometry revealed that the majority of proteins that undergo age-related changes in CSF of either mouse model were linked to microglia and astrocytes. Specifically, we identified a panel of more than 20 glial-derived proteins that were increased in CSF of aged ß-amyloid precursor protein- and α-synuclein-transgenic mice and largely overlap with previously described disease-associated glial genes identified by single-cell transcriptomics. Our results also show that enhanced shedding is responsible for the increase of several of the identified glial CSF proteins as exemplified for TREM2. Notably, the vast majority of these proteins can also be quantified in human CSF and reveal changes in Alzheimer's disease cohorts. The finding that cellular transcriptome changes translate into corresponding changes of CSF proteins is of clinical relevance, supporting efforts to identify fluid biomarkers that reflect the various functional states of glial responses in cerebral proteopathies, such as Alzheimer's and Parkinson's disease.
Asunto(s)
Enfermedad de Alzheimer , Líquido Cefalorraquídeo , Neuroglía , Enfermedad de Parkinson , Proteoma , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/metabolismo , Animales , Biomarcadores/líquido cefalorraquídeo , Líquido Cefalorraquídeo/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Neuroglía/metabolismo , Enfermedad de Parkinson/líquido cefalorraquídeo , Enfermedad de Parkinson/metabolismo , Proteoma/metabolismo , Análisis de la Célula Individual , Proteínas tauRESUMEN
Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18 h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and "outside-in" directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.
Asunto(s)
Mitofagia , Ubiquitina-Proteína Ligasas , Células HeLa , Humanos , Fosforilación , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Trans-activation response DNA binding protein of 43 kDa (TDP-43) regulates a great variety of cellular processes in the nucleus and cytosol. In addition, a defined subset of neurodegenerative diseases is characterized by nuclear depletion of TDP-43 as well as cytosolic mislocalization and aggregation. To perform its diverse functions TDP-43 can associate with different ribonucleoprotein complexes. Combined with transcriptomics, MS interactome studies have unveiled associations between TDP-43 and the spliceosome machinery, polysomes and RNA granules. Moreover, the highly dynamic, low-valency interactions regulated by its low-complexity domain calls for innovative proximity labeling methodologies. In addition to protein partners, the analysis of post-translational modifications showed that they may play a role in the nucleocytoplasmic shuttling, RNA binding, liquid-liquid phase separation and protein aggregation of TDP-43. Here we review the various TDP-43 ribonucleoprotein complexes characterized so far, how they contribute to the diverse functions of TDP-43, and roles of post-translational modifications. Further understanding of the fluid dynamic properties of TDP-43 in ribonucleoprotein complexes, RNA granules, and self-assemblies will advance the understanding of RNA processing in cells and perhaps help to develop novel therapeutic approaches for TDPopathies.
Asunto(s)
Agregado de Proteínas , Proteómica , Proteínas de Unión al ADN/genética , RibonucleoproteínasRESUMEN
Mitochondria are important organelles in eukaryotes. Turnover and quality control of mitochondria are regulated at the transcriptional and posttranslational level by several cellular mechanisms. Removal of defective mitochondrial proteins is mediated by mitochondria resident proteases or by proteasomal degradation of individual proteins. Clearance of bulk mitochondria occurs via a selective form of autophagy termed mitophagy. In yeast and some developing metazoan cells (e.g., oocytes and reticulocytes), mitochondria are largely removed by ubiquitin-independent mechanisms. In such cases, the regulation of mitophagy is mediated via phosphorylation of mitochondria-anchored autophagy receptors. On the other hand, ubiquitin-dependent recruitment of cytosolic autophagy receptors occurs in situations of cellular stress or disease, where dysfunctional mitochondria would cause oxidative damage. In mammalian cells, a well-studied ubiquitin-dependent mitophagy pathway induced by mitochondrial depolarization is regulated by the mitochondrial protein kinase PINK1, which upon activation recruits the ubiquitin ligase parkin. Here, we review mechanisms of mitophagy with an emphasis on posttranslational modifications that regulate various mitophagy pathways. We describe the autophagy components involved with particular emphasis on posttranslational modifications. We detail the phosphorylations mediated by PINK1 and parkin-mediated ubiquitylations of mitochondrial proteins that can be modulated by deubiquitylating enzymes. We also discuss the role of accessory factors regulating mitochondrial fission/fusion and the interplay with pro- and antiapoptotic Bcl-2 family members. Comprehensive knowledge of the processes of mitophagy is essential for the understanding of vital mitochondrial turnover in health and disease.
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Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Mitofagia , Transducción de Señal , Ubiquitinación , Animales , Mitocondrias/genética , Proteínas Mitocondriales/genéticaRESUMEN
Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress-induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R-like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal-regulated protein kinase modules and glycogen synthase kinase 3ß. For arsenite, but not for sorbitol, quenching oxidative stress with N-acetylcysteine did suppress both SG formation and TDP-43 ubiquitylation and insolubility. Thus, sodium arsenite appears to promote SG formation and TDP-43 modifications via oxidative stress, but sorbitol stimulates TDP-43 ubiquitylation and insolubility via a novel pathway(s) independent of SG formation. In conclusion, pathological TDP-43 modifications can be mediated via multiple distinct pathways for which SGs are not essential.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Choque Térmico/genética , Estrés Oxidativo/genética , Ubiquitinación/genética , Acetilcisteína/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Proteínas de Unión al ADN/química , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Glucógeno Sintasa Quinasa 3 beta/genética , Células HEK293 , Proteínas de Choque Térmico/química , Humanos , Indoles/farmacología , Mutación/efectos de los fármacos , Presión Osmótica/efectos de los fármacos , Agregación Patológica de Proteínas/genética , Transporte de Proteínas/genética , Transducción de Señal/efectos de los fármacos , Solubilidad/efectos de los fármacos , Sorbitol/farmacologíaRESUMEN
The pathophysiology of Parkinson's disease is characterized by the abnormal accumulation of α-synuclein (α-Syn), eventually resulting in the formation of Lewy bodies and neurites in surviving neurons in the brain. Although α-Syn aggregation has been extensively studied in vitro, there is limited in vivo knowledge on α-Syn aggregation. Here, we used the powerful genetics of Drosophila melanogaster and developed an in vivo assay to monitor α-Syn accumulation by using a bimolecular fluorescence complementation assay. We found that both genetic and pharmacologic manipulations affected α-Syn accumulation. Interestingly, we also found that alterations in the cellular protein degradation mechanisms strongly influenced α-Syn accumulation. Administration of compounds identified as risk factors for Parkinson's disease, such as rotenone or heavy metal ions, had only mild or even no impact on α-Syn accumulation in vivo. Finally, we show that increasing phosphorylation of α-Syn at serine 129 enhances the accumulation and toxicity of α-Syn. Altogether, our study establishes a novel model to study α-Syn accumulation and illustrates the complexity of manipulating proteostasis in vivo.-Prasad, V., Wasser, Y., Hans, F., Goswami, A., Katona, I., Outeiro, T. F., Kahle, P. J., Schulz, J. B., Voigt, A. Monitoring α-synuclein multimerization in vivo.
Asunto(s)
Amiloide/química , Modelos Animales de Enfermedad , Drosophila melanogaster/metabolismo , Multimerización de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Animales , Drosophila melanogaster/crecimiento & desarrollo , Masculino , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Serina , alfa-Sinucleína/genéticaRESUMEN
Parkin is an ubiquitin ligase regulating mitochondrial quality control reactions, including the autophagic removal of depolarized mitochondria (mitophagy). Parkin-mediated protein ubiquitinations may be counteracted by deubiquitinating enzymes (DUBs). We conducted a high-content imaging screen of Parkin translocation to depolarized mitochondria after siRNA mediated silencing of each DUB in Parkin overexpressing HeLa cells. Knockdown of the ubiquitin-specific protease USP36 led to delayed Parkin translocation while only slightly disturbing the ubiquitination of mitochondrial proteins, but final autophagic elimination of mitochondria was severely disrupted. The localization of the nucleolar USP36 was not altered during mitophagy. However, the marker for transcriptional active chromatin, histone 2B Lys120 mono-ubiquitination was found reduced in USP36-silenced cells undergoing mitophagy. We observed a reduction of the mRNA and protein levels of Beclin-1 and its associated autophagy-related key regulator ATG14L in USP36 knockdown cells. Importantly, transfection of active ATG14L into USP36-silenced cells significantly restored Parkin-dependent mitophagy. We propose USP36 as regulator for the Parkin-dependent mitophagy at least in part via the Beclin-1-ATG14L pathway.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Beclina-1/genética , Regulación hacia Abajo/genética , Mitofagia/genética , Ubiquitina Tiolesterasa/genética , Ubiquitina-Proteína Ligasas/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen/métodos , Células HeLa , Humanos , Mitocondrias/genética , Proteínas Mitocondriales/genética , Ubiquitina/genética , Proteasas Ubiquitina-Específicas/genética , Ubiquitinación/genéticaRESUMEN
TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, particularly in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. A pathognomonic TDP-43 C-terminal fragment (CTF) spanning amino acids 193-414 contains only four lysine residues that could be potentially ubiquitinylated. Here, serial mutagenesis of these four lysines to arginine revealed that not a single residue is responsible for the ubiquitinylation of mCherry-tagged CTF. Removal of all four lysines was necessary to suppress ubiquitinylation. Interestingly, Lys-408 substitution enhanced the pathological phosphorylation of the immediately adjacent serine residues 409/410 in the context of mCherry-CTF. Thus, Lys-408 ubiquitinylation appears to hinder Ser-409/410 phosphorylation in TDP-43 CTF. However, we did not observe the same effect for full-length TDP-43. We extended the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were identified in the nuclear localization sequence (NLS; Lys-84 and Lys-95) and RNA-binding region (mostly Lys-160, Lys-181, and Lys-263). Mutagenesis of Lys-84 confirmed its importance as the major determinant for nuclear import, whereas Lys-95 mutagenesis did not significantly affect TDP-43's nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant had significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites indicates that the NLS residues Lys-84 and Lys-95 have more prominent roles in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Ubiquitina/metabolismo , Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Lisina/química , Mutagénesis Sitio-Dirigida , Mutación , Fosforilación , Procesamiento Proteico-Postraduccional , SolubilidadRESUMEN
Site-directed A-to-I RNA editing is a technology for re-programming genetic information at the RNA-level. We describe here the first design of genetically encodable guideRNAs that enable the re-addressing of human ADAR2 toward specific sites in user-defined mRNA targets. Up to 65% editing yield has been achieved in cell culture for the recoding of a premature Stop codon (UAG) into tryptophan (UIG). In the targeted gene, editing was very specific. We applied the technology to recode a recessive loss-of-function mutation in PINK1 (W437X) in HeLa cells and showed functional rescue of PINK1/Parkin-mediated mitophagy, which is linked to the etiology of Parkinson's disease. In contrast to other editing strategies, this approach requires no artificial protein. Our novel guideRNAs may allow for the development of a platform technology that requires only the administration or expression of a guideRNA to recode genetic information, with high potential for application in biology and medicine.
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Adenosina Desaminasa/metabolismo , Mitofagia , Mutación Puntual , Proteínas Quinasas/genética , Edición de ARN , Proteínas de Unión al ARN/metabolismo , Codón sin Sentido , Genoma , Células HEK293 , Células HeLa , Humanos , ARN Guía de Kinetoplastida/química , ARN Mensajero/metabolismo , TransfecciónRESUMEN
Leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein, is a key causative factor in Parkinson's disease (PD). Identification of novel substrates and the molecular mechanisms underlying the effects of LRRK2 are essential for understanding the pathogenesis of PD. In this study, we showed that LRRK2 played an important role in neuronal cell death by directly phosphorylating and activating apoptosis signal-regulating kinase 1 (ASK1). LRRK2 phosphorylated ASK1 at Thr832 that is adjacent to Thr845, which serves as an autophosphorylation site. Moreover, results of binding and kinase assays showed that LRRK2 acted as a scaffolding protein by interacting with each components of the ASK1-MKK3/6-p38 MAPK pathway through its specific domains and increasing the proximity to downstream targets. Furthermore, LRRK2-induced apoptosis was suppressed by ASK1 inhibition in neuronal stem cells derived from patients with PD. These results clearly indicate that LRRK2 acts as an upstream kinase in the ASK1 pathway and plays an important role in the pathogenesis of PD.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , MAP Quinasa Quinasa Quinasa 5/genética , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Apoptosis/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Neuronas/patología , Enfermedad de Parkinson/patología , Fosforilación , Transducción de Señal/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
The abnormal accumulation of α-synuclein aggregates in neurons, nerve fibers, or glial cells is the hallmark of a group of neurodegenerative diseases known collectively as α-synucleinopathies. Clinical, neuropathological, and experimental evidence strongly suggests that α-synuclein plays a role not only as a trigger of pathological processes at disease inception, but also as a mediator of pathological spreading during disease progression. Specific properties of α-synuclein, such as its ability to pass from one neuron to another, its tendency to aggregate, and its potential to generate self-propagating species, have been described and elucidated in animal models and may contribute to the relentless exacerbation of Parkinson's disease pathology in patients. Animal models used for studying α-synuclein accumulation, aggregation, and propagation are mostly based on three approaches: (1) intra-parenchymal inoculations of exogenous α-synuclein (e.g., synthetic α-synuclein fibrils), (2) transgenic mice, and (3) animals (mice or rats) in which α-synuclein overexpression is induced by viral vector injections. Whereas pathological α-synuclein changes are consistently observed in these models, important differences are also found. In particular, pronounced pathology in transgenic mice and viral vector-injected animals does not appear to involve self-propagating α-synuclein species. A critical discussion of these models reveals their strengths and limitations and provides the basis for recommendations concerning their use for future investigations.
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alfa-Sinucleína/metabolismo , Animales , Animales Modificados Genéticamente , Modelos Animales de Enfermedad , Vectores Genéticos/metabolismo , HumanosRESUMEN
α-Synuclein (αSYN) is the neuropathological hallmark protein of Parkinson's disease (PD) and related neurodegenerative disorders. Moreover, the gene encoding αSYN (SNCA) is a major genetic contributor to PD. Interestingly, independent genome-wide association studies also identified SNCA as the most important candidate gene for alcoholism. Furthermore, single-nucleotide-polymorphisms have been associated with alcohol-craving behavior and alcohol-craving patients showed augmented αSYN expression in blood. To investigate the effect of αSYN on the addictive properties of chronic alcohol use, we examined consumption, motivation, and seeking responses induced by environmental stimuli and relapse behavior in transgenic mice expressing the human mutant [A30P]αSYN throughout the brain. The primary reinforcing effects of alcohol under operant self-administration conditions were increased, while consumption and the alcohol deprivation effect were not altered in the transgenic mice. The same mice were subjected to immunohistochemical measurements of immediate-early gene inductions in brain regions involved in addiction-related behaviors. Acute ethanol injection enhanced immunostaining for the phosphorylated form of cAMP response element binding protein in both amygdala and nucleus accumbens of αSYN transgenic mice, while in wild-type mice no effect was visible. However, at the same time, levels of cFos remain unchanged in both genotypes. These results provide experimental confirmation of SNCA as a candidate gene for alcoholism in addition to its known link to PD.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Motivación/efectos de los fármacos , Motivación/genética , alfa-Sinucleína/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Depresores del Sistema Nervioso Central/sangre , Conducta de Elección/efectos de los fármacos , Señales (Psicología) , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Etanol/sangre , Extinción Psicológica/efectos de los fármacos , Preferencias Alimentarias/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Locomoción/efectos de los fármacos , Locomoción/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Autoadministración , Gusto/efectos de los fármacos , Gusto/genética , alfa-Sinucleína/genéticaRESUMEN
Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal deposits observed in Parkinson's disease and dementia with Lewy bodies. The objective of the study was to identify surface-exposed epitopes of alpha-synuclein in vitro and in vivo formed aggregates. Polyclonal immunoglobulin Y antibodies were raised against short linear peptides of the alpha-synuclein molecule. An epitope in the N-terminal region (1-10) and all C-terminal epitopes (90-140) were found to be exposed in an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant monomeric, oligomeric, and fibrillar alpha-synuclein. In a phospholipid ELISA, the N-terminus and mid-region of alpha-synuclein (i.e., 1-90) were associated with phosphatidylserine and thus occluded from antibody binding. The antibodies that reacted most strongly with epitopes in the in vitro aggregates (i.e., 1-10 and epitopes between positions 90-140) also labeled alpha-synuclein inclusions in brains from transgenic (Thy-1)-h[A30P] alpha-synuclein mice and Lewy bodies and Lewy neurites in brains of patients with alpha-synucleinopathies. However, differences in reactivity were observed with the C-terminal antibodies when brain tissue from human and transgenic mice was compared. Taken together, the study shows that although similar epitopes are exposed in both in vitro and in vivo formed alpha-synuclein inclusions, structural heterogeneity can be observed between different molecular species.
Asunto(s)
Mapeo Cromosómico/métodos , Epítopos/genética , Epítopos/metabolismo , Agregado de Proteínas/fisiología , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Anciano de 80 o más Años , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana EdadRESUMEN
We provide evidence that S6 kinase 1 (S6K1) Aly/REF-like target (SKAR) is engaged in IFN-α signaling and plays a key role in the generation of IFN responses. Our data demonstrate that IFN-α induces phosphorylation of SKAR, which is mediated by either the p90 ribosomal protein S6 kinase (RSK) or p70 S6 kinase (S6K1), in a cell type-specific manner. This type I IFN-inducible phosphorylation of SKAR results in enhanced interaction with the eukaryotic initiation factor (eIF)4G and recruitment of activated RSK1 to 5' cap mRNA. Our studies also establish that SKAR is present in cap-binding CBP80 immune complexes and that this interaction is mediated by eIF4G. We demonstrate that inducible protein expression of key IFN-α-regulated protein products such as ISG15 and p21(WAF1/CIP1) requires SKAR activity. Importantly, our studies define a requirement for SKAR in the generation of IFN-α-dependent inhibitory effects on malignant hematopoietic progenitors from patients with chronic myeloid leukemia or myeloproliferative neoplasms. Taken altogether, these findings establish critical and essential roles for SKAR in the regulation of mRNA translation of IFN-sensitive genes and induction of IFN-α biological responses.
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Interferón-alfa/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citocinas/metabolismo , Guanosina/análogos & derivados , Guanosina/metabolismo , Humanos , Ratones , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Ubiquitinas/metabolismoRESUMEN
Lewy bodies and neurites are the pathological hallmark of Parkinson's disease. These structures are composed of fibrillized and ubiquitinated alpha-synuclein suggesting that impaired protein clearance is an important event in aggregate formation. The A30P mutation is known for its fast oligomerization, but slow fibrillization rate. Despite its toxicity to neurons, mechanisms involved in either clearance or conversion of A30P alpha-synuclein from its soluble state into insoluble fibrils and their effects in vivo are poorly understood. Synphilin-1 is present in Lewy bodies, interacting with alpha-synuclein in vivo and in vitro and promotes its sequestration into aggresomes, which are thought to act as cytoprotective agents facilitating protein degradation. We therefore crossed animals overexpressing A30P alpha-synuclein with synphilin-1 transgenic mice to analyze its impact on aggregation, protein clearance and phenotype progression. We observed that co-expression of synphilin-1 mildly delayed the motor phenotype caused by A30P alpha-synuclein. Additionally, the presence of N- and C-terminal truncated alpha-synuclein species and fibrils were strongly reduced in double-transgenic mice when compared with single-transgenic A30P mice. Insolubility of mutant A30P and formation of aggresomes was still detectable in aged double-transgenic mice, paralleled by an increase of ubiquitinated proteins and high autophagic activity. Hence, this study supports the notion that co-expression of synphilin-1 promotes formation of autophagic-susceptible aggresomes and consecutively the degradation of human A30P alpha-synuclein. Notably, although synphilin-1 overexpression significantly reduced formation of fibrils and astrogliosis in aged animals, a similar phenotype is present in single- and double-transgenic mice suggesting additional neurotoxic processes in disease progression.
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Proteínas Portadoras/genética , Proteínas del Tejido Nervioso/genética , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Envejecimiento , Animales , Autofagia/fisiología , Benzotiazoles , Encéfalo/metabolismo , Encéfalo/patología , Proteínas Portadoras/metabolismo , Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Enfermedad de Parkinson/genética , Pliegue de Proteína , Solubilidad , Tiazoles/metabolismo , Ubiquitina/metabolismoRESUMEN
Lewy bodies, a pathological hallmark of Parkinson's disease (PD), contain aggregated alpha-synuclein (αSyn), which is found in several modified forms and can be discovered phosphorylated, ubiquitinated and truncated. Aggregation-prone truncated species of αSyn caused by aberrant cleavage of this fibrillogenic protein are hypothesized to participate in its sequestration into inclusions subsequently leading to synaptic dysfunction and neuronal death. Here, we investigated the role of calpain cleavage of αSyn in vivo by generating two opposing mouse models. We crossed into human [A30P]αSyn transgenic (i) mice deficient for calpastatin, a calpain-specific inhibitor, thus enhancing calpain activity (SynCAST(-)) and (ii) mice overexpressing human calpastatin leading to reduced calpain activity (SynCAST(+)). As anticipated, a reduced calpain activity led to a decreased number of αSyn-positive aggregates, whereas loss of calpastatin led to increased truncation of αSyn in SynCAST(-). Furthermore, overexpression of calpastatin decreased astrogliosis and the calpain-dependent degradation of synaptic proteins, potentially ameliorating the observed neuropathology in [A30P]αSyn and SynCAST(+) mice. Overall, our data further support a crucial role of calpains, particularly of calpain 1, in the pathogenesis of PD and in disease-associated aggregation of αSyn, indicating a therapeutic potential of calpain inhibition in PD.
Asunto(s)
Proteínas de Unión al Calcio/genética , Calpaína/genética , Enfermedad de Parkinson/genética , Agregación Patológica de Proteínas/genética , alfa-Sinucleína/genética , Animales , Proteínas de Unión al Calcio/metabolismo , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Agregado de Proteínas , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Proteolisis , Transducción de Señal , Sinapsis/metabolismo , Sinapsis/patología , alfa-Sinucleína/metabolismoRESUMEN
Depolarized mitochondria are degraded by mitophagy in a process that depends on the Parkinson's disease gene products PINK1 and Parkin. This is accompanied by ubiquitylation of several mitochondrial substrates. The roles of E2 ubiquitin-conjugating enzymes (UBE2) in mitophagy are poorly understood. Here, we investigate a set of UBE2 enzymes that might regulate Parkin-mediated mitophagy. Knockdown of the E2 enzymes UBE2N, UBE2L3 or UBE2D2 and UBE2D3 (UBE2D2/3) significantly reduced autophagic clearance of depolarized mitochondria. However, this did not interfere with mitochondrial PINK1 stabilization and Parkin translocation. UBE2N knockdown prevented specifically K63-linked ubiquitylation at mitochondrial sites. Nevertheless, polyubiquitin and p62 (officially known as SQSTM1) were still found on mitochondria after individual UBE2 knockdown. Knockdown of all of these UBE2s together significantly reduced mitochondrial polyubiquitylation and p62 recruitment. Moreover, reduced ubiquitylation of mitofusins, the mitochondrial import receptor subunits TOM20 and TOM70, the voltage-dependent anion channel protein 1 and Parkin was observed in cells silenced for all of these UBE2s. A version of Parkin with a mutation in the active site (C431S) failed to ubiquitylate these mitochondrial substrates even in the presence of UBE2s. We conclude that UBE2N, UBE2L3 and UBE2D2/3 synergistically contribute to Parkin-mediated mitophagy.
Asunto(s)
Mitocondrias/fisiología , Enfermedad de Parkinson/enzimología , Proteínas Quinasas/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Células HeLa , Homeostasis , Humanos , Potencial de la Membrana Mitocondrial , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mitofagia/genética , Mutación/genética , Enfermedad de Parkinson/genética , ARN Interferente Pequeño/genética , Receptores de Superficie Celular/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
Trans-activation element DNA-binding protein of 43 kDa (TDP-43) characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors found in a yeast two-hybrid screen using an adult human brain cDNA library. We confirmed the TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase Y (UBPY). When cells were treated with proteasome inhibitor, ubiquitinated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitination of TDP-43, whereas catalytically inactive UBE2E3(C145S) was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitination. We examined 15 of the 48 known disease-associated TDP-43 mutants and found that one was excessively ubiquitinated. This strong TDP-43(K263E) ubiquitination was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced TDP-43(K263E) ubiquitination. Moreover, wild-type but not active site mutant UBPY reduced ubiquitination of TDP-43 C-terminal fragments and of a nuclear import-impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitination, solubility, and neurodegeneration.