RESUMO
Large cytosolic protein aggregates are removed by two main cellular processes, autophagy and the ubiquitin-proteasome system, and defective clearance of these protein aggregates results in proteotoxicity and cell death. Recently, we found that the eIF2α kinase heme-regulated inhibitory (HRI) induced a cytosolic unfolded protein response to prevent aggregation of innate immune signalosomes, but whether HRI acts as a general sensor of proteotoxicity in the cytosol remains unclear. Here we show that HRI controls autophagy to clear cytosolic protein aggregates when the ubiquitin-proteasome system is inhibited. We further report that silencing the expression of HRI resulted in decreased levels of BAG3 and HSPB8, two proteins involved in chaperone-assisted selective autophagy, suggesting that HRI may control proteostasis in the cytosol at least in part through chaperone-assisted selective autophagy. Moreover, knocking down the expression of HRI resulted in cytotoxic accumulation of overexpressed α-synuclein, a protein known to aggregate in Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. In agreement with these data, protein aggregate accumulation and microglia activation were observed in the spinal cord white matter of 7-month-old Hri-/- mice as compared with Hri+/+ littermates. Moreover, aged Hri-/- mice showed accumulation of misfolded α-synuclein in the lateral collateral pathway, a region of the sacral spinal cord horn that receives visceral sensory afferents from the bladder and distal colon, a pathological feature common to α-synucleinopathies in humans. Together, these results suggest that HRI contributes to a general cytosolic unfolded protein response that could be leveraged to bolster the clearance of cytotoxic protein aggregates.
Assuntos
Autofagia , Microglia/metabolismo , Agregados Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Medula Espinal/metabolismo , Resposta a Proteínas não Dobradas , eIF-2 Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Células HEK293 , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Camundongos , Camundongos Knockout , Microglia/patologia , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Medula Espinal/patologia , eIF-2 Quinase/genéticaRESUMO
BACKGROUND: SNCA mutations cause autosomal dominant parkinsonism and inform our understanding of the molecular underpinnings of synucleinopathies. The most recently identified mutation, p.Ala53Glu (A53E), has only been observed in Finland. The objectives of this study were to examine clinical, genetic, epigenetic, and biochemical features of the first family outside Finland with A53E. METHODS: We examined a Canadian family with parkinsonism because of A53E using haplotype and DNA methylation analyses. We assessed aggregation properties of A53E α-synuclein in vitro. RESULTS: Family members with parkinsonism shared a common haplotype distinct from Finnish patients with A53E. Increased acceleration of DNA methylation age was accompanied by earlier age at onset in the family members. We demonstrate that A53E α-synuclein has a propensity to form oligomers and phosphorylation promotes fibrillation. CONCLUSIONS: A53E as a cause of parkinsonism is not restricted to Finnish individuals. DNA methylation may contribute to disease age at onset. A53E enriches α-synuclein oligomers and fibrils dependent on the phosphorylation state. © 2018 International Parkinson and Movement Disorder Society.
Assuntos
Expressão Gênica/genética , Mutação/genética , Transtornos Parkinsonianos/genética , alfa-Sinucleína/genética , Idoso , Epigenômica/métodos , Feminino , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , LinhagemRESUMO
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.
Assuntos
Loci Gênicos/genética , Predisposição Genética para Doença/genética , Complexos Multiproteicos/metabolismo , Doença de Parkinson/enzimologia , Mapeamento de Interação de Proteínas/métodos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Análise de Variância , Western Blotting , Encéfalo/metabolismo , Fracionamento Celular , Primers do DNA/genética , Estudo de Associação Genômica Ampla/métodos , Complexo de Golgi/ultraestrutura , Células HEK293 , Humanos , Imunoprecipitação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Espectrometria de Massas , Microscopia Confocal , Complexos Multiproteicos/genética , Plasmídeos/genética , Proteínas Serina-Treonina Quinases/genética , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Parkinson's disease (PD) is a progressive neurogenerative movement disorder characterized by dopaminergic cell death within the substantia nigra pars compacta (SNpc) due to the aggregation-prone protein α-synuclein. Accumulation of α-synuclein is implicated in mitochondrial dysfunction and disruption of the autophagic turnover of mitochondria, or mitophagy, which is an essential quality control mechanism proposed to preserve mitochondrial fidelity in response to aging and stress. Yet, the precise relationship between α-synuclein accumulation, mitochondrial autophagy, and dopaminergic cell loss remains unresolved. Here, we determine the kinetics of α-synuclein overexpression and mitophagy using the pH-sensitive fluorescent mito-QC reporter. We find that overexpression of mutant A53T α-synuclein in either human SH-SY5Y cells or rat primary cortical neurons induces mitophagy. Moreover, the accumulation of mutant A53T α-synuclein in the SNpc of rats results in mitophagy dysregulation that precedes the onset of dopaminergic neurodegeneration. This study reveals a role for mutant A53T α-synuclein in inducing mitochondrial dysfunction, which may be an early event contributing to neurodegeneration.
RESUMO
Accumulation of α-synuclein into toxic oligomers or fibrils is implicated in dopaminergic neurodegeneration in Parkinson's disease. Here we performed a high-throughput, proteome-wide peptide screen to identify protein-protein interaction inhibitors that reduce α-synuclein oligomer levels and their associated cytotoxicity. We find that the most potent peptide inhibitor disrupts the direct interaction between the C-terminal region of α-synuclein and CHarged Multivesicular body Protein 2B (CHMP2B), a component of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III). We show that α-synuclein impedes endolysosomal activity via this interaction, thereby inhibiting its own degradation. Conversely, the peptide inhibitor restores endolysosomal function and thereby decreases α-synuclein levels in multiple models, including female and male human cells harboring disease-causing α-synuclein mutations. Furthermore, the peptide inhibitor protects dopaminergic neurons from α-synuclein-mediated degeneration in hermaphroditic C. elegans and preclinical Parkinson's disease models using female rats. Thus, the α-synuclein-CHMP2B interaction is a potential therapeutic target for neurodegenerative disorders.
Assuntos
Doença de Parkinson , Masculino , Feminino , Animais , Ratos , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Caenorhabditis elegans/metabolismo , Neurônios Dopaminérgicos/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismoRESUMO
TNFα stimulation triggers both cell death and survival programs. Since dysregulated apoptosis or cell growth can cause inflammatory diseases, cancer, or autoimmune disorders, it is important to understand the molecular mechanism of controlling cell death and survival by TNFR downstream signaling molecules. In this study, we used normal diploid cells, mouse embryonic fibroblasts (MEFs), to mimic the general TNFα-resistant phenomenon seen under physiological conditions.We elucidated the TNFα-induced death signaling complexes in TNF α-resistant WT MEFs and TNFα-sensitive MEFs that were cFLIP-, RelA-, TRAF2- or RIP1-deficient. Consistent with TNFα-mediated killing, we detected TNFα-induced high molecular weight complexes containing caspase-8 and FADD by gel filtration in the deficient MEFs, especially in those devoid of cFLIP. In addition to the presence of caspase-8-FADD in the TNFα-induced-death complex in the deficient MEFs, we also detected an intermediate protein complex containing RIP1, TRAF2 and caspase-8.Moreover, we demonstrated a correlation between TNFα-sensitivity and death-inducing complex ability in two transformed cell lines, E1A- and Ras- transformed MEFs and PDGF-B-transformed NIH-3T3 cells with PDGF-B signaling inhibited by the tyrosine kinase inhibitor STI571. Taken together, our results suggest the involvement of cFLIP-, RelA-, RIP1-, or TRAF2-related mechanisms for preventing FADD-caspase-8 interaction in wild-type MEFs.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/deficiência , Caspase 8/metabolismo , Morte Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Proteína de Domínio de Morte Associada a Fas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas Ativadoras de GTPase/deficiência , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Células NIH 3T3 , Fator 2 Associado a Receptor de TNF/deficiência , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Fas-associated death domain (FADD) and caspase-8 are key signal transducers for death receptor-induced apoptosis, whereas cellular FLICE-inhibitory protein (cFLIP) antagonizes this process. Interestingly, FADD and caspase-8 also play a role in T cell development and T cell receptor (TCR)-mediated proliferative responses. To investigate the underlying mechanism, we generated cFLIP-deficient T cells by reconstituting Rag-/- blastocysts with cFLIP-deficient embryonic stem cells. These Rag chimeric mutant mice (rcFLIP-/-) had severely reduced numbers of T cells in the thymus, lymph nodes, and spleen, although mature T lymphocytes did develop. Similar to FADD- or caspase-8-deficient cells, rcFLIP-/- T cells were impaired in proliferation in response to TCR stimulation. Further investigation revealed that cFLIP is required for T cell survival, as well as T cell cycling in response to TCR stimulation. Interestingly, some signaling pathways from the TCR complex appeared competent, as CD3 plus CD28 cross-linking was capable of activating the ERK pathway in rcFLIP-/- T cells. We demonstrate an essential role for cFLIP in T cell function.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Linfócitos T/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Caspase 8 , Caspases/metabolismo , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteína de Domínio de Morte Associada a Fas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Tumor necrosis factor receptor 1-associated death domain protein (TRADD) is the core adaptor recruited to TNF receptor 1 (TNFR1) upon TNFalpha stimulation. In cells from TRADD-deficient mice, TNFalpha-mediated apoptosis and TNFalpha-stimulated NF-kappaB, JNK, and ERK activation are defective. TRADD is also important for germinal center formation, DR3-mediated costimulation of T cells, and TNFalpha-mediated inflammatory responses in vivo. TRADD deficiency does not enhance IFNgamma-induced signaling. Importantly, TRADD has a novel role in TLR3 and TLR4 signaling. TRADD participates in the TLR4 complex formed upon LPS stimulation, and TRADD-deficient macrophages show impaired cytokine production in response to TLR ligands in vitro. Thus, TRADD is a multifunctional protein crucial both for TNFR1 signaling and other signaling pathways relevant to immune responses.
Assuntos
Transdução de Sinais , Proteína de Domínio de Morte Associada a Receptor de TNF/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Apoptose , Centro Germinativo , Inflamação , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Linfócitos T , Proteína de Domínio de Morte Associada a Receptor de TNF/fisiologia , Receptor 3 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Parkinson's disease is a disabling neurodegenerative movement disorder characterized by dopaminergic neuron loss induced by α-synuclein oligomers. There is an urgent need for disease-modifying therapies for Parkinson's disease, but drug discovery is challenged by lack of in vivo models that recapitulate early stages of neurodegeneration. Invertebrate organisms, such as the nematode worm Caenorhabditis elegans, provide in vivo models of human disease processes that can be instrumental for initial pharmacological studies. METHODS: To identify early motor impairment of animals expressing α-synuclein in dopaminergic neurons, we first used a custom-built tracking microscope that captures locomotion of single C. elegans with high spatial and temporal resolution. Next, we devised a method for semi-automated and blinded quantification of motor impairment for a population of simultaneously recorded animals with multi-worm tracking and custom image processing. We then used genetic and pharmacological methods to define the features of early motor dysfunction of α-synuclein-expressing C. elegans. Finally, we applied the C. elegans model to a drug repurposing screen by combining it with an artificial intelligence platform and cell culture system to identify small molecules that inhibit α-synuclein oligomers. Screen hits were validated using in vitro and in vivo mammalian models. RESULTS: We found a previously undescribed motor phenotype in transgenic α-synuclein C. elegans that correlates with mutant or wild-type α-synuclein protein levels and results from dopaminergic neuron dysfunction, but precedes neuronal loss. Together with artificial intelligence-driven in silico and in vitro screening, this C. elegans model identified five compounds that reduced motor dysfunction induced by α-synuclein. Three of these compounds also decreased α-synuclein oligomers in mammalian neurons, including rifabutin which has not been previously investigated for Parkinson's disease. We found that treatment with rifabutin reduced nigrostriatal dopaminergic neurodegeneration due to α-synuclein in a rat model. CONCLUSIONS: We identified a C. elegans locomotor abnormality due to dopaminergic neuron dysfunction that models early α-synuclein-mediated neurodegeneration. Our innovative approach applying this in vivo model to a multi-step drug repurposing screen, with artificial intelligence-driven in silico and in vitro methods, resulted in the discovery of at least one drug that may be repurposed as a disease-modifying therapy for Parkinson's disease.
Assuntos
Transtornos Motores , alfa-Sinucleína , Animais , Inteligência Artificial , Caenorhabditis elegans/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Mamíferos/metabolismo , Transtornos Motores/metabolismo , Ratos , alfa-Sinucleína/metabolismoRESUMO
Molecular chaperones are critical to maintaining intracellular proteostasis and have been shown to have a protective role against alpha-synuclein-mediated toxicity. Co-chaperone proteins regulate the activity of molecular chaperones and connect the chaperone network to protein degradation and cell death pathways. Bcl-2 associated athanogene 5 (BAG5) is a co-chaperone that modulates proteostasis by inhibiting the activity of Heat shock protein 70 (Hsp70) and several E3 ubiquitin ligases, resulting in enhanced neurodegeneration in models of Parkinson's disease (PD). Here we identify a novel interaction between BAG5 and p62/sequestosome-1 (SQSTM1), suggesting that BAG5 may bridge the chaperone network to autophagy-mediated protein degradation. We found that BAG5 enhanced the formation of pathogenic alpha-synuclein oligomers and regulated the levels and subcellular distribution of p62. These results extend the role of BAG5 in alpha-synuclein processing and intracellular proteostasis.
RESUMO
Parkinson's disease is a progressive neurodegenerative disorder characterised by the accumulation of misfolded α-synuclein in selected brain regions, including the substantia nigra pars compacta (SNpc), where marked loss of dopaminergic neurons is also observed. Yet, the relationship between misfolded α-synuclein and neurotoxicity currently remains unclear. As the principal route for degradation of misfolded proteins in mammalian cells, the ubiquitin-proteasome system (UPS) is critical for maintenance of cellular proteostasis. Misfolded α-synuclein impairs UPS function and contributes to neuronal death in vitro. Here, we examine its effects in vivo using adeno-associated viruses to co-express A53T α-synuclein and the ubiquitinated reporter protein UbG76V-GFP in rat SNpc. We found that α-synuclein over-expression leads to early-onset catalytic impairment of the 26S proteasome with associated UPS dysfunction, preceding the onset of behavioural deficits and dopaminergic neurodegeneration. UPS failure in dopaminergic neurons was also associated with selective accumulation of α-synuclein phosphorylated at the serine 129 residue, which has previously been linked to increased neurotoxicity. Our study highlights a role for α-synuclein in disturbing proteostasis which may contribute to neurodegeneration in vivo.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Parte Compacta da Substância Negra/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismo , Animais , Feminino , Células HEK293 , Humanos , Ratos Sprague-DawleyRESUMO
As pathogenic Parkin mutations result in the defective clearance of damaged mitochondria, Parkin-dependent mitophagy is thought to be protective against the dopaminergic neurodegeneration observed in Parkinson's disease. Recent studies, however, have demonstrated that Parkin can promote cell death in the context of severe mitochondrial damage by degrading the pro-survival Bcl-2 family member, Mcl-1. Therefore, Parkin may act as a 'switch' that can shift the balance between protective or pro-death pathways depending on the degree of mitochondrial damage. Here, we report that the Parkin interacting protein, Bcl-2-associated athanogene 5 (BAG5), impairs mitophagy by suppressing Parkin recruitment to damaged mitochondria and reducing the movement of damaged mitochondria into the lysosomes. BAG5 also enhanced Parkin-mediated Mcl-1 degradation and cell death following severe mitochondrial insult. These results suggest that BAG5 may regulate the bi-modal activity of Parkin, promoting cell death by suppressing Parkin-dependent mitophagy and enhancing Parkin-mediated Mcl-1 degradation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose , Mitofagia , Ubiquitina-Proteína Ligases/metabolismo , Apoptose/efeitos dos fármacos , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Modelos Biológicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacosRESUMO
Tumor necrosis factor (TNF) induces both cell death and survival signals. NF-kappaB, a transcription factor activated by TNF, is critical for controlling survival signals through trans-activation of downstream target genes. However, few NF-kappaB target survival genes have been identified with direct roles in oncogenesis. We report that platelet-derived growth factor B (PDGF-B), an oncogene and growth factor, is highly induced by TNF in fibroblasts in an NF-kappaB-dependent manner. PDGF-B can rescue NF-kappaB-deficient fibroblasts from TNF-mediated killing, and inhibition of PDGF-B signaling sensitizes wild-type cells to TNF-induced death. Interestingly, PDGF-B-transformed NIH-3T3 cells are even more highly sensitized to TNF-induced cell death with PDGF-B inhibition. Our results suggest that while normal cells contain multiple TNF-induced survival signals, tumor cells may favor a specific survival gene that is abnormally upregulated in order to evade death signals.
Assuntos
Proteínas Proto-Oncogênicas c-sis/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Células 3T3 , Animais , Apoptose , Northern Blotting , Western Blotting , Caspase 3 , Caspases/metabolismo , Morte Celular , Linhagem Celular Transformada , Separação Celular , Sobrevivência Celular , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Fibroblastos/metabolismo , Citometria de Fluxo , Quinase I-kappa B , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Células NIH 3T3 , Proteínas Serina-Treonina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , Regulação para CimaRESUMO
BACKGROUND: The forniceal area is currently being evaluated as a target for deep brain stimulation (DBS) to improve cognitive function in patients with Alzheimer's disease. The molecular changes at downstream targets within the stimulated circuit are unknown. OBJECTIVE: To analyze the modulation of hippocampal protein expression following 1 h of fornix DBS in the rat. METHODS: Animals underwent bilateral forniceal DBS for 1 h and sacrificed at different time-points after the initiation of the stimulation (1 h, 2.5 h, 5 h, 25 h). Bilateral hippocampi were isolated for western blot analyses. RESULTS: Forniceal DBS led to a dramatic elevation of cFos post-stimulation, suggesting that forniceal DBS activates the hippocampus. There was also a significant increase in candidate proteins including several trophic factors, such as brain derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) but not glial cell-derived neurotrophic factor (GDNF). There was in addition, increased expression of the synaptic markers growth associated protein 43 (GAP-43), synaptophysin and α-synuclein. No changes were observed at the studied time-points in Alzheimer's-related proteins including amyloid precursor protein (APP), tau, phosphorylated tau (ptau), or selected chaperone proteins (HSP40, HSP70 and CHIP). CONCLUSIONS: Forniceal DBS triggers hippocampal activity and rapidly modulate the expression of neurotrophic factors and markers of synaptic plasticity known to play key roles in memory processing. The clinical effects of DBS of the fornix may, in part, be mediated by producing changes in the expression of these proteins.
Assuntos
Estimulação Encefálica Profunda/métodos , Fórnice/metabolismo , Hipocampo/metabolismo , Fatores de Crescimento Neural/biossíntese , Biossíntese de Proteínas/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Cognição/fisiologia , Masculino , Plasticidade Neuronal/fisiologia , Ratos , Ratos Wistar , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Mice homozygous for the disrupted renal type IIa sodium/phosphate (Na/Pi) cotransporter gene (Npt2-/-) exhibit renal Pi wasting, hypophosphatemia, and an adaptive increase in the serum concentration of 1,25-dihydroxyvitamin D with associated hypercalcemia and hypercalciuria. Because hypercalciuria is a risk factor for nephrocalcinosis, we determined whether Npt2-/- mice form renal stones. Analysis of renal sections by von Kossa staining and intact kidneys by microcomputed tomography revealed renal calcification in adult Npt2-/- mice but not in Npt2+/+ littermates. Energy-dispersive spectroscopy and selected-area electron diffraction indicated that the calcifications are comprised of calcium and Pi with an apatitic mineral phase. To determine the age of onset of nephrocalcinosis, we examined renal sections of newborn and weanling mice. At both ages, mutant but not wild-type mice display renal calcification, which is associated with renal Pi wasting and hypercalciuria. Immunohistochemistry revealed that osteopontin co-localizes with the calcifications. Furthermore, renal osteopontin messenger RNA abundance is significantly elevated in Npt2-/- mice compared with Npt2+/+ mice. The onset of renal stones correlated developmentally with the absence of Npt2 expression and the expression of the genes responsible for the renal production (1alpha-hydroxylase) and catabolism (24-hydroxylase) of 1,25-dihydroxyvitamin D. In summary, we show that Npt2 gene ablation is associated with renal calcification and suggest that mutations in the NPT2 gene may contribute to nephrocalcinosis in a subset of patients with familial hypercalciuria.
Assuntos
Cálculos Renais/genética , Simportadores/deficiência , Simportadores/genética , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Sequência de Bases , Cálcio/urina , Sistema Enzimático do Citocromo P-450/genética , DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Hipercalcemia/genética , Hipercalcemia/metabolismo , Rim/metabolismo , Cálculos Renais/metabolismo , Cálculos Renais/patologia , Camundongos , Camundongos Knockout , Mutação , Fosfatos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo I , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIa , Esteroide Hidroxilases/genética , Simportadores/metabolismo , Vitamina D3 24-HidroxilaseRESUMO
Parkinson's disease (PD) is a common neurodegenerative condition in which abnormalities in protein homeostasis, or proteostasis, may lead to accumulation of the protein α-synuclein (α-syn). Mutations within or multiplications of the gene encoding α-syn are known to cause genetic forms of PD and polymorphisms in the gene are recently established risk factors for idiopathic PD. α-syn is a major component of Lewy bodies, the intracellular proteinaceous inclusions which are pathological hallmarks of most forms of PD. Recent evidence demonstrates that α-syn can self associate into soluble oligomeric species and implicates these α-syn oligomers in cell death. We have previously shown that carboxyl terminus of Hsp70-interacting protein (CHIP), a co-chaperone molecule with E3 ubiquitin ligase activity, may reduce the levels of toxic α-syn oligomers. Here we demonstrate that α-syn is ubiquitinylated by CHIP both in vitro and in cells. We find that the products from ubiquitinylation by CHIP include both monoubiquitinylated and polyubiquitinylated forms of α-syn. We also demonstrate that CHIP and α-syn exist within a protein complex with the co-chaperone bcl-2-associated athanogene 5 (BAG5) in brain. The interaction of CHIP with BAG5 is mediated by Hsp70 which binds to the tetratricopeptide repeat domain of CHIP and the BAG domains of BAG5. The Hsp70-mediated association of BAG5 with CHIP results in inhibition of CHIP E3 ubiquitin ligase activity and subsequently reduces α-syn ubiquitinylation. Furthermore, we use a luciferase-based protein-fragment complementation assay of α-syn oligomerization to investigate regulation of α-syn oligomers by CHIP in living cells. We demonstrate that BAG5 mitigates the ability of CHIP to reduce α-syn oligomerization and that non-ubiquitinylated α-syn has an increased propensity for oligomerization. Thus, our results identify CHIP as an E3 ubiquitin ligase of α-syn and suggest a novel function for BAG5 as a modulator of CHIP E3 ubiquitin ligase activity with implications for CHIP-mediated regulation of α-syn oligomerization.
Assuntos
Proteínas de Transporte/fisiologia , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Idoso de 80 Anos ou mais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Domínio Catalítico/genética , Domínio Catalítico/fisiologia , Feminino , Humanos , Luciferases/genética , Luciferases/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Multimerização Proteica , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Transfecção , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologia , UbiquitinaçãoRESUMO
Disruption of the major renal Na-phosphate (Pi) cotransporter gene Npt2a in mice leads to a substantial decrease in renal brush-border membrane Na-Pi cotransport, hypophosphatemia, and appropriate adaptive increases in renal 25-hydroxyvitamin D3-1alpha-hydroxylase (1alphaOHase) activity and the serum concentration of 1,25-dihydroxyvitamin D3 [1,25(OH)2D]. The latter is associated with increased intestinal Ca absorption, hypercalcemia, hypercalciuria, and renal calcification in Npt2-/- mice. To determine the contribution of elevated serum 1,25(OH)2D levels to the development of hypercalciuria and nephrocalcinosis in Npt2-/- mice, we examined the effects of 1alphaOHase gene ablation and long-term Pi supplementation on urinary Ca excretion and renal calcification by microcomputed tomography. We show that the urinary Ca/creatinine ratio is significantly decreased in Npt2-/-/1alphaOHase-/- mice compared with Npt2-/- mice. In addition, renal calcification, determined by estimating the calcified volume to total renal volume (CV/TV), is reduced by 80% in Npt2-/-/1alphaOHase-/- mice compared with that in Npt2-/- mice. In Npt2-/- mice derived from dams fed a 1% Pi diet and maintained on the same diet, we observed a significant decrease in urinary Ca/creatinine that was also associated with 80% reduction in CV/TV when compared with counterparts fed a 0.6% diet. Taken together, the present data demonstrate that both 1alphaOHase gene ablation and Pi supplementation inhibit renal calcification in Npt2-/- mice and that 1,25(OH)2D is essential for the development of hypercalciuria and nephrocalcinosis in the mutant strain.
Assuntos
25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Nefrocalcinose/fisiopatologia , Simportadores/genética , Animais , Calcinose/fisiopatologia , Calcitriol/sangue , Cálcio/sangue , Cálcio/urina , Feminino , Homozigoto , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Nefrocalcinose/patologia , Fósforo na Dieta/farmacologia , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo I , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIIRESUMO
Dementia of the Alzheimer type (DAT) is common in older persons with Down syndrome (DS). There are three common alleles of the apolipoprotein E (ApoE) gene (Sigma 2, Sigma 3, and Sigma 4) resulting in three different isoforms (E2, E3, and E4) and six different genotypes (2,2; 2,3; 2,4; 3,3; 3,4; and 4,4). Sigma 4 is a risk factor for DAT whereas Sigma 2 appears prophylactic. As hepatitis B virus (HBV) infection and hypothyroidism also are common in DS, we evaluated associations between ApoE type, HBV status, and thyroid status in a sample of older persons with DS (n = 55; mean age, 44.3 +/- 10.8 years) using chi-squared analysis. Participants were classified as E2 (2,2 or 2,3), E3 (3,3), or E4 (3,4 or 4,4); positive for markers of HBV infection in the present or past (i.e., total HBcAb+ and/or HBsAg+ with or without infectivity, defined as HBV+) or negative for markers of HBV infection (defined as HBV-) and, currently receiving thyroid hormone supplement (defined as "hypothyroidism") or having normal thyroid function. The majority of the HBV+ were currently HBcAb+ and HBsAb+, but not HBsAg+. In females, there was an ApoE allele effect on thyroid status (P < or = 0.01), E2 being negatively (P < or = 0.01) and E4 being positively (P < or = 0.05) associated with "hypothyroidism". There was no evidence for an ApoE allele effect on thyroid status in males. There was no evidence for an ApoE allele effect on HBV status, or for an HBV status effect on thyroid status. As thyroid status can affect cognitive function, ApoE allele effects in DAT may, in part, be thyroid effects.