RESUMO
Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS.
Assuntos
Esclerose Lateral Amiotrófica , Animais , Camundongos , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Cisteína/genética , Mutação , Superóxido Dismutase/genética , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that share pathological features, including the aberrant accumulation of ubiquitinated protein inclusions within motor neurons. Previously, we have shown that the sequestration of ubiquitin (Ub) into inclusions disrupts Ub homeostasis in cells expressing ALS-associated variants superoxide dismutase 1 (SOD1), fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43). Here, we investigated whether an ALS/FTD-linked pathogenic variant in the CCNF gene, encoding the E3 Ub ligase Cyclin F (CCNF), also perturbs Ub homeostasis. The presence of a pathogenic CCNF variant was shown to cause ubiquitin-proteasome system (UPS) dysfunction in induced pluripotent stem cell-derived motor neurons harboring the CCNF S621G mutation. The expression of the CCNFS621G variant was associated with an increased abundance of ubiquitinated proteins and significant changes in the ubiquitination of key UPS components. To further investigate the mechanisms responsible for this UPS dysfunction, we overexpressed CCNF in NSC-34 cells and found that the overexpression of both wild-type (WT) and the pathogenic variant of CCNF (CCNFS621G) altered free Ub levels. Furthermore, double mutants designed to decrease the ability of CCNF to form an active E3 Ub ligase complex significantly improved UPS function in cells expressing both CCNFWT and the CCNFS621G variant and were associated with increased levels of free monomeric Ub. Collectively, these results suggest that alterations to the ligase activity of the CCNF complex and the subsequent disruption to Ub homeostasis play an important role in the pathogenesis of CCNF-associated ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Doença de Pick , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Ciclinas/genética , Neurônios Motores/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Doença de Pick/metabolismo , Homeostase/genética , MutaçãoRESUMO
A growing wave of evidence has placed the concept of protein homeostasis at the center of the pathogenesis of amyotrophic lateral sclerosis (ALS). This is due primarily to the presence of pathological transactive response DNA-binding protein (TDP-43), fused in sarcoma (FUS) or superoxide dismutase-1 (SOD1) inclusions within motor neurons of ALS postmortem tissue. However, the earliest pathological alterations associated with ALS occur to the structure and function of the synapse, prior to motor neuron loss. Recent evidence demonstrates the pathological accumulation of ALS-associated proteins (TDP-43, FUS, C9orf72-associated di-peptide repeats and SOD1) within the axo-synaptic compartment of motor neurons. In this review, we discuss this recent evidence and how axo-synaptic proteome dyshomeostasis may contribute to synaptic dysfunction in ALS.
RESUMO
Mutant superoxide dismutase 1 (SOD1) can be constitutively released from motor neurons and transmitted to naïve motor neurons to promote the progression of amyotrophic lateral sclerosis (ALS). However, the biological impacts of this process and the precise mechanisms of SOD1 release remain to be fully resolved. Using biochemical and fluorescent techniques, this study aimed to determine if P2X7 receptor activation could induce mutant SOD1 release from motor neurons and whether this released SOD1 could be transmitted to motor neurons or microglia to mediate effects associated with neurodegeneration in ALS. Aggregated SOD1G93A, released from murine NSC-34 motor neurons transiently transfected with SOD1G93A, could be transmitted to naïve NSC-34 cells and murine EOC13 microglia to induce endoplasmic reticulum (ER) stress and tumour necrosis factor-alpha (TNFα) release, respectively. Immunoblotting revealed NSC-34 cells expressed P2X7. Extracellular ATP induced cation dye uptake into these cells, which was blocked by the P2X7 antagonist AZ10606120, demonstrating these cells express functional P2X7. Moreover, ATP induced the rapid release of aggregated SOD1G93A from NSC-34 cells transiently transfected with SOD1G93A, a process blocked by AZ10606120 and revealing a role for P2X7 in this process. ATP-induced SOD1G93A release coincided with membrane blebbing. Finally, aggregated SOD1G93A released via P2X7 activation could also be transmitted to NSC-34 and EOC13 cells to induce ER stress and TNFα release, respectively. Collectively, these results identify a novel role for P2X7 in the prion-like propagation of SOD1 in ALS and provide a possible explanation for the therapeutic benefits of P2X7 antagonism previously observed in ALS SOD1G93A mice.
Assuntos
Esclerose Lateral Amiotrófica , Receptores Purinérgicos P2X7 , Superóxido Dismutase-1 , Animais , Camundongos , Trifosfato de Adenosina/farmacologia , Esclerose Lateral Amiotrófica/patologia , Modelos Animais de Doenças , Camundongos Transgênicos , Neurônios Motores/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Superóxido Dismutase-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Protein aggregates that result in inclusion formation are a pathological hallmark common to many neurodegenerative diseases, including amyotrophic lateral sclerosis, Parkinson's disease and Huntington's disease. Under conditions of cellular stress, activation of the heat shock response (HSR) results in an increase in the levels of molecular chaperones and is a first line of cellular defence against inclusion formation. It remains to be established whether neurodegenerative disease-associated proteins and inclusions are themselves capable of inducing an HSR in neuronal cells. To address this, we generated a neuroblastoma cell line that expresses a fluorescent reporter protein under conditions of heat shock transcription factor 1 (HSF1)-mediated HSR induction. We show that the HSR is not induced by exogenous treatment with aggregated forms of recombinant α-synuclein or the G93A mutant of superoxide dismutase-1 (SOD1G93A) nor intracellular expression of SOD1G93A or a pathogenic form of polyglutamine-expanded huntingtin (Htt72Q). These results suggest that pathogenic proteins evade detection or impair induction of the HSR in neuronal cells. A failure of protein aggregation to induce an HSR might contribute to the development of inclusion pathology in neurodegenerative diseases.This article has an associated First Person interview with the first author of the paper.
Assuntos
Doenças Neurodegenerativas , Fatores de Transcrição de Choque Térmico/genética , Resposta ao Choque Térmico/genética , Humanos , Doenças Neurodegenerativas/genética , Agregados Proteicos , Superóxido Dismutase-1RESUMO
The discovery that prion protein can misfold into a pathological conformation that encodes structural information capable of both propagation and inducing severe neuropathology has revolutionized our understanding of neurodegenerative disease. Many neurodegenerative diseases with a protein misfolding component are now classified as "prion-like" owing to the propagation of both symptoms and protein aggregation pathology in affected individuals. The neuromuscular disorder amyotrophic lateral sclerosis (ALS) is characterized by protein inclusions formed by either TAR DNA-binding protein of 43 kDa (TDP-43), Cu/Zn superoxide dismutase (SOD1), or fused in sarcoma (FUS), in both upper and lower motor neurons. Evidence from in vitro, cell culture, and in vivo studies has provided strong evidence to support the involvement of a prion-like mechanism in ALS. In this article, we review the evidence suggesting that prion-like propagation of protein aggregation is a primary pathomechanism in ALS, focusing on the key proteins and genes involved in disease (TDP-43, SOD1, FUS, and C9orf72). In each case, we discuss the evidence ranging from biophysical studies to in vivo examinations of prion-like spreading. We suggest that the idiopathic nature of ALS may stem from its prion-like nature and that elucidation of the specific propagating protein assemblies is paramount to developing effective therapies.
RESUMO
TAR DNA-binding protein 43 (TDP-43) has been identified as the major constituent of the proteinaceous inclusions that are characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin positive frontotemporal lobar degeneration (FTLD). Wild type TDP-43 inclusions are a pathological hallmark of >95% of patients with sporadic ALS and of the majority of familial ALS cases, and they are also found in a significant proportion of FTLD cases. ALS is the most common form of motor neuron disease, characterized by progressive weakness and muscular wasting, and typically leads to death within a few years of diagnosis. To determine how the translocation and misfolding of TDP-43 contribute to ALS pathogenicity, it is crucial to define the dynamic behavior of this protein within the cellular environment. It is therefore necessary to develop cell models that allow the location of the protein to be defined. We report the use of TDP-43 with a tetracysteine tag for visualization using fluorogenic biarsenical compounds and show that this model displays features of ALS observed in other cell models. We also demonstrate that this labeling procedure enables live-cell imaging of the translocation of the protein from the nucleus into the cytosol.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Cisteína/química , Proteínas de Ligação a DNA/metabolismo , Fluoresceínas/química , Modelos Biológicos , Compostos Organometálicos/química , Sitios de Sequências Rotuladas , Sequência de Aminoácidos , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citosol/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Eletroporação , Corantes Fluorescentes , Degeneração Lobar Frontotemporal/metabolismo , Humanos , Cinética , Imagem Óptica , Transporte Proteico , Imagem com Lapso de Tempo , TransfecçãoRESUMO
A hallmark of amyotrophic lateral sclerosis (ALS) pathology is the accumulation of ubiquitylated protein inclusions within motor neurons. Recent studies suggest the sequestration of ubiquitin (Ub) into inclusions reduces the availability of free Ub, which is essential for cellular function and survival. However, the dynamics of the Ub landscape in ALS have not yet been described. Here, we show that Ub homeostasis is altered in a cell model of ALS induced by expressing mutant SOD1 (SOD1A4V). By monitoring the distribution of Ub in cells expressing SOD1A4V, we show that Ub is present at the earliest stages of SOD1A4V aggregation, and that cells containing SOD1A4V aggregates have greater ubiquitin-proteasome system (UPS) dysfunction. Furthermore, SOD1A4V aggregation is associated with the redistribution of Ub and depletion of the free Ub pool. Ubiquitomics analysis indicates that expression of SOD1A4V is associated with a shift of Ub to a pool of supersaturated proteins, including those associated with oxidative phosphorylation and metabolism, corresponding with altered mitochondrial morphology and function. Taken together, these results suggest that misfolded SOD1 contributes to UPS dysfunction and that Ub homeostasis is an important target for monitoring pathological changes in ALS.This article has an associated First Person interview with the first author of the paper.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Homeostase , Superóxido Dismutase-1/metabolismo , Ubiquitina/metabolismo , Esclerose Lateral Amiotrófica/etiologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Mutação , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Superóxido Dismutase-1/genéticaRESUMO
Superoxide dismutase-1 (SOD1) mutants, including those with unaltered enzymatic activity, are known to cause amyotrophic lateral sclerosis (ALS). Several destabilizing factors contribute to pathogenicity including a reduced ability to complete the normal maturation process which comprises folding, metal cofactor acquisition, intra-subunit disulphide bond formation and dimerization. Immature SOD1 forms toxic oligomers and characteristic large insoluble aggregates within motor system cells. Here we report that the cysteine-reactive molecule ebselen efficiently confers the SOD1 intra-subunit disulphide and directs correct SOD1 folding, depopulating the globally unfolded precursor associated with aggregation and toxicity. Assisted formation of the unusual SOD1 cytosolic disulphide bond could have potential therapeutic applications. In less reducing environments, ebselen forms a selenylsulphide with Cys111 and restores the monomer-dimer equilibrium of A4V SOD1 to wild-type. Ebselen is therefore a potent bifunctional pharmacological chaperone for SOD1 that combines properties of the SOD1 chaperone hCCS and the recently licenced antioxidant drug, edaravone.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Antioxidantes/farmacologia , Azóis/farmacologia , Compostos Organosselênicos/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Superóxido Dismutase-1/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Antioxidantes/uso terapêutico , Asma/tratamento farmacológico , Asma/patologia , Azóis/uso terapêutico , Cristalografia por Raios X , Cisteína/química , Dissulfetos/química , Edaravone/farmacologia , Células HEK293 , Humanos , Isoindóis , Chaperonas Moleculares/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Compostos Organosselênicos/uso terapêutico , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genéticaRESUMO
It is now widely accepted in the field that the normally secreted chaperone clusterin is redirected to the cytosol during endoplasmic reticulum (ER) stress, although the physiological function(s) of this physical relocation remain unknown. We have examined in this study whether or not increased expression of clusterin is able to protect neuronal cells against intracellular protein aggregation and cytotoxicity, characteristics that are strongly implicated in a range of neurodegenerative diseases. We used the amyotrophic lateral sclerosis-associated protein TDP-43 as a primary model to investigate the effects of clusterin on protein aggregation and neurotoxicity in complementary in vitro, neuronal cell and Drosophila systems. We have shown that clusterin directly interacts with TDP-43 in vitro and potently inhibits its aggregation, and observed that in ER stressed neuronal cells, clusterin co-localized with TDP-43 and specifically reduced the numbers of cytoplasmic inclusions. We further showed that the expression of TDP-43 in transgenic Drosophila neurons induced ER stress and that co-expression of clusterin resulted in a dramatic clearance of mislocalized TDP-43 from motor neuron axons, partially rescued locomotor activity and significantly extended lifespan. We also showed that in Drosophila photoreceptor cells, clusterin co-expression gave ER stress-dependent protection against proteotoxicity arising from both Huntingtin-Q128 and mutant (R406W) human tau. We therefore conclude that increased expression of clusterin can provide an important defense against intracellular proteotoxicity under conditions that mimic specific features of neurodegenerative disease.
Assuntos
Clusterina/metabolismo , Clusterina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Síndromes Neurotóxicas/tratamento farmacológico , Animais , Animais Geneticamente Modificados , Linhagem Celular Tumoral , Clusterina/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Estresse do Retículo Endoplasmático/genética , Olho/metabolismo , Olho/ultraestrutura , Hemolinfa/citologia , Humanos , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Larva , Atividade Motora/genética , Atividade Motora/fisiologia , Neurônios Motores/ultraestrutura , Neuroblastoma/patologia , Síndromes Neurotóxicas/genética , Síndromes Neurotóxicas/patologia , Agregados Proteicos/efeitos dos fármacos , Agregados Proteicos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3-5 years of diagnosis. Recently, we identified disease-linked mutations in the CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) (Williams KL et al 2016 Nat. Commun.7, 11253. (doi:10.1038/ncomms11253)). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST (proline, glutamic acid, serine and threonine) sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF(cyclin F) complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF(cyclin F) E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.
Assuntos
Caseína Quinase II/metabolismo , Ciclinas/metabolismo , Complexos Multiproteicos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida , Ativação Enzimática , Células HEK293 , Humanos , Lisina , Espectrometria de Massas , Modelos Moleculares , Fosfatidilserinas , Fosforilação , Ligação Proteica , UbiquitinaçãoRESUMO
Amyotrophic lateral sclerosis is a devastating neuromuscular degenerative disease characterized by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology including TAR DNA-binding protein of 43 kDa (TDP-43) aggregates. Previous work suggests that TDP-43 can move between cells. Here we used a novel flow cytometry technique (FloIT) to analyze TDP-43 inclusions and propagation. When cells were transfected to express either mutant G294A TDP-43 fused to GFP or wild type TDP-43fused to tomato red and then co-cultured, flow cytometry detected intact cells containing both fusion proteins and using FloIT detected an increase in the numbers of inclusions in lysates from cells expressing wild type TDP-43-tomato. Furthermore, in this same model, FloIT analyses detected inclusions containing both fusion proteins. These results imply the transfer of TDP-43 fusion proteins between cells and that this process can increase aggregation of wild-type TDP-43 by a mechanism involving co-aggregation with G294A TDP-43.
Assuntos
Proteínas de Ligação a DNA/química , Citometria de Fluxo/métodos , Corpos de Inclusão/química , Proteínas Mutantes/química , Agregados Proteicos , Animais , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Corpos de Inclusão/metabolismo , Camundongos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that disease-affected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting.
Assuntos
Esclerose Lateral Amiotrófica/fisiopatologia , Agregação Patológica de Proteínas/fisiopatologia , Nervos Espinhais/fisiopatologia , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Encéfalo/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/fisiologia , Neurônios Motores/metabolismo , Mutação , Agregados Proteicos/fisiologia , Agregação Patológica de Proteínas/metabolismo , Dobramento de Proteína , Proteína FUS de Ligação a RNA/metabolismo , Medula Espinal/metabolismo , Nervos Espinhais/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1/genéticaRESUMO
Amyotrophic lateral sclerosis is a rapidly progressing neurodegenerative disease associated with protein misfolding and aggregation. Most cases are characterized by TDP-43 positive inclusions, while a minority of familial ALS cases are instead FUS and SOD1 positive respectively. Cells can generate inclusions of variable type including previously characterized aggresomes, IPOD or JUNQ structures depending on the misfolded protein. SOD1 invariably forms JUNQ inclusions but it remains unclear whether other ALS protein aggregates arise as one of these previously described inclusion types or form unique structures. Here we show that FUS variably partitioned to IPOD, JUNQ or alternate structures, contain a mobile fraction, were not microtubule dependent and initially did not contain ubiquitin. TDP-43 inclusions formed in a microtubule independent manner, did not contain a mobile fraction but variably colocalized to JUNQ inclusions and another alternate structure. We conclude that the RNA binding proteins TDP-43 and FUS do not consistently fit the currently characterised inclusion models suggesting that cells have a larger repertoire for generating inclusions than currently thought, and imply that toxicity in ALS does not stem from a particular aggregation process or aggregate structure.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Corpos de Inclusão/metabolismo , Proteínas Mutantes/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Superóxido Dismutase/metabolismo , Linhagem Celular Tumoral , Humanos , Microtúbulos/metabolismo , Agregados Proteicos , Especificidade por Substrato , Transfecção , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
SerpinB2 (PAI-2), a member of the clade B family of serine protease inhibitors, is one of the most upregulated proteins following cellular stress. Originally described as an inhibitor of urokinase plasminogen activator, its predominant cytoplasmic localisation suggests an intracellular function. SerpinB2 has been reported to display cytoprotective properties in neurons and to interact with intracellular proteins including components of the ubiquitin-proteasome system (UPS). In the current study we explored the potential role of SerpinB2 as a modulator of proteotoxic stress. Initially, we transiently transfected wild-type SerpinB2 and SerpinB2-/- murine embryonic fibroblasts (MEFs) with Huntingtin exon1-polyglutamine (fused C-terminally to mCherry). Inclusion body formation as result of Huntingtin aggregation was evident in the SerpinB2 expressing cells but significantly impaired in the SerpinB2-/- cells, the latter concomitant with loss in cell viability. Importantly, recovery of the wild-type phenotype and cell viability was rescued by retroviral transduction of SerpinB2 expression. SerpinB2 modestly attenuated Huntingtin and amyloid beta fibril formation in vitro and was able to bind preferentially to misfolded proteins. Given the modest chaperone-like activity of SerpinB2 we tested the ability of SerpinB2 to modulate UPS and autophagy activity using a GFP reporter system and autophagy reporter, respectively. Activity of the UPS was reduced and autophagy was dysregulated in SerpinB2-/- compared to wild-type MEFs. Moreover, we observed a non-covalent interaction between ubiquitin and SerpinB2 in cells using GFP-pulldown assays and bimolecular fluorescence complementation. We conclude that SerpinB2 plays an important role in proteostasis as its loss leads to a proteotoxic phenotype associated with an inability to compartmentalize aggregating proteins and a reduced capacity of the UPS.
Assuntos
Citoproteção/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Inibidor 2 de Ativador de Plasminogênio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Peptídeos beta-Amiloides/química , Animais , Éxons/genética , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Peptídeos/metabolismo , Gravidez , Agregados Proteicos , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Ubiquitina/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterised by the progressive degeneration of brain and spinal cord motor neurons. Ubiquitin-proteasome system (UPS) dysfunction and oxidative stress have been implicated in ALS pathogenesis. However, it is unknown whether the defects in these pathways extend to non-neuronal tissues such as fibroblasts. Fibroblasts, unlike neuronal tissue, are readily available and may hold potential for short-term, rapid diagnostic and prognostic purposes. We investigated whether primary skin fibroblasts from ALS patients share, or can be manipulated to develop, functional and pathological abnormalities seen in affected neuronal cells. We inhibited UPS function and induced oxidative stress in the fibroblasts and found that ALS-related cellular changes, such as aggregate formation and ubiquitination of ALS-associated proteins (TDP-43 and ubiquilin 2), can be reproduced in these cells. Higher levels of TDP-43 ubiquitination, as evident by colocalization between TDP-43 and ubiquitin, were found in all six ALS cases compared to controls following extracellular insults. In contrast, colocalization between ubiquilin 2 and ubiquitin was not markedly different between ALS cases and control. A UPS reporter assay revealed UPS abnormalities in patient fibroblasts. Despite the presence of ALS-related cellular changes in the patient fibroblasts, no elevated toxicity was observed. This suggests that aggregate formation and colocalization of ALS-associated proteins may be insufficient alone to confer toxicity in fibroblasts used in the present study. Chronic exposure to ALS-linked stresses and the ALS-linked cellular pathologies may be necessary to breach an unknown threshold that triggers cell death.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Fibroblastos/patologia , Fibroblastos/fisiologia , Pele/patologia , Pele/fisiopatologia , Proteínas Adaptadoras de Transdução de Sinal , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/cirurgia , Proteínas Relacionadas à Autofagia , Biópsia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Imunofluorescência , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Mutação , Estresse Oxidativo/fisiologia , Transfecção , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively "misfolded" state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.
Assuntos
Peptídeos/química , Dobramento de Proteína , Superóxido Dismutase/química , Amiloide/química , Humanos , Corpos de Inclusão/química , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Peptídeos/genética , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
Dissociation of superoxide dismutase 1 dimers is enhanced by glutathionylation, although the dissociation constants reported to date are imprecise. We have quantified the discreet dissociation constants for wild-type superoxide dismutase 1 and six naturally occurring sequence variants, in their unmodified and glutathionylated forms, at the ratios expressed. Unmodified superoxide dismutase 1 variants that shared similar dissociation constants with SOD1(WT) had disproportionately increased dissociation constants when glutathionylated. This defines a key role for glutathionylation in superoxide dismutase 1 associated familial amyotrophic lateral sclerosis.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/genética , Glutationa/metabolismo , Humanos , Cinética , Espectrometria de Massas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutase/química , Superóxido Dismutase/genética , Superóxido Dismutase-1RESUMO
The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5'-triphosphate (ATP) and 2'(3')-O-(4-benzoylbenzoyl) ATP induced ethidium(+) or YO-PRO-1(2+) uptake into both cell lines. ATP induced ethidium(+) uptake into EOC13 cells in a concentration-dependent manner, with an EC(50) of ~130 µM. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75-100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca(2+) influx and K(+) efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia.
Assuntos
Morte Celular/efeitos dos fármacos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Animais , Linhagem Celular , Immunoblotting , Camundongos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetrazóis/farmacologia , Tiazóis/farmacologiaRESUMO
α(2)-Macroglobulin (α(2)M) is an extracellular chaperone that inhibits amorphous and fibrillar protein aggregation. The reaction of α(2)M with proteases results in an 'activated' conformation, where the proteases become covalently-linked within the interior of a cage-like structure formed by α(2)M. This study investigates, the effect of activation on the ability of α(2)M to inhibit amyloid formation by Aß(1-42) and I59T human lysozyme and shows that protease-activated α(2)M can act via two distinct mechanisms: (i) by trapping proteases that remain able to degrade polypeptide chains and (ii) by a chaperone action that prevents misfolded clients from continuing along the amyloid forming pathway.