RESUMO
UBQLN2 mutations cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD), but the pathogenic mechanisms by which they cause disease remain unclear. Proteomic profiling identified 'mitochondrial proteins' as comprising the largest category of protein changes in the spinal cord (SC) of the P497S UBQLN2 mouse model of ALS/FTD. Immunoblots confirmed P497S animals have global changes in proteins predictive of a severe decline in mitochondrial health, including oxidative phosphorylation (OXPHOS), mitochondrial protein import and network dynamics. Functional studies confirmed mitochondria purified from the SC of P497S animals have age-dependent decline in nearly all steps of OXPHOS. Mitochondria cristae deformities were evident in spinal motor neurons of aged P497S animals. Knockout (KO) of UBQLN2 in HeLa cells resulted in changes in mitochondrial proteins and OXPHOS activity similar to those seen in the SC. KO of UBQLN2 also compromised targeting and processing of the mitochondrial import factor, TIMM44, resulting in accumulation in abnormal foci. The functional OXPHOS deficits and TIMM44-targeting defects were rescued by reexpression of WT UBQLN2 but not by ALS/FTD mutant UBQLN2 proteins. In vitro binding assays revealed ALS/FTD mutant UBQLN2 proteins bind weaker with TIMM44 than WT UBQLN2 protein, suggesting that the loss of UBQLN2 binding may underlie the import and/or delivery defect of TIMM44 to mitochondria. Our studies indicate a potential key pathogenic disturbance in mitochondrial health caused by UBQLN2 mutations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/genética , Proteínas Relacionadas à Autofagia/genética , Demência Frontotemporal/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutação , Animais , Linhagem Celular , Modelos Animais de Doenças , Células HeLa , Humanos , Immunoblotting , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Consumo de Oxigênio/genética , Proteômica/métodosRESUMO
Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS UBQLN2 patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat UBQLN2-linked ALS/FTD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Autofagossomos/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia/genética , Demência/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas Relacionadas à Autofagia/genética , Biomarcadores/metabolismo , Linhagem Celular , Demência/metabolismo , Demência/patologia , Predisposição Genética para Doença , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Ligação Proteica , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/genética , Proteínas Relacionadas à Autofagia/genética , Demência Frontotemporal/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteômica/métodos , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/metabolismo , Linhagem Celular , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Serotonina/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Aggregation of misfolded proteins or peptides is a common feature of neurodegenerative diseases including Alzheimer's, Parkinson's, Huntington's, prion and other diseases. Recent years have witnessed a growing number of reports of overlap in neuropathological features that were once thought to be unique to only one neurodegenerative disorder. However, the origin for the overlap remains unclear. One possibility is that diseases with mixed brain pathologies might arise from cross-seeding of one amyloidogenic protein by aggregated states of unrelated proteins. In the current study we examined whether prion replication can be induced by cross-seeding by α-synuclein or Aß peptide. We found that α-synuclein aggregates formed in cultured cells or in vitro display cross-seeding activity and trigger misfolding of the prion protein (PrPC) in serial Protein Misfolding Cyclic Amplification reactions, producing self-replicating PrP states characterized by a short C-terminal proteinase K (PK)-resistant region referred to as PrPres. Non-fibrillar α-synuclein or fibrillar Aß failed to cross-seed misfolding of PrPC. Remarkably, PrPres triggered by aggregated α-synuclein in vitro propagated in animals and, upon serial transmission, produced PrPSc and clinical prion disease characterized by spongiosis and astrocytic gliosis. The current study demonstrates that aggregated α-synuclein is potent in cross-seeding of prion protein misfolding and aggregation in vitro, producing self-replicating states that can lead to transmissible prion diseases upon serial passaging in wild type animals. In summary, the current work documents direct cross-seeding between unrelated amyloidogenic proteins associated with different neurodegenerative diseases. This study suggests that early interaction between unrelated amyloidogenic proteins might underlie the etiology of mixed neurodegenerative proteinopathies.
Assuntos
Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Príons/metabolismo , alfa-Sinucleína/metabolismo , Animais , Cricetinae , Humanos , Mesocricetus , Camundongos , Dobramento de ProteínaRESUMO
Missense mutations in ubiquilin 2 (UBQLN2) cause ALS with frontotemporal dementia (ALS-FTD). Animal models of ALS are useful for understanding the mechanisms of pathogenesis and for preclinical investigations. However, previous rodent models carrying UBQLN2 mutations failed to manifest any sign of motor neuron disease. Here, we show that lines of mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have cognitive deficits, shortened lifespans, and develop motor neuron disease, mimicking the human disease. Neuropathologic analysis of the mice with end-stage disease revealed the accumulation of ubiquitinated inclusions in the brain and spinal cord, astrocytosis, a reduction in the number of hippocampal neurons, and reduced staining of TAR-DNA binding protein 43 in the nucleus, with concomitant formation of ubiquitin+ inclusions in the cytoplasm of spinal motor neurons. Moreover, both lines displayed denervation muscle atrophy and age-dependent loss of motor neurons that correlated with a reduction in the number of large-caliber axons. By contrast, two mouse lines expressing WT UBQLN2 were mostly devoid of clinical and pathological signs of disease. These UBQLN2 mouse models provide valuable tools for identifying the mechanisms underlying ALS-FTD pathogenesis and for investigating therapeutic strategies to halt disease.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/genética , Mutação de Sentido Incorreto , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Esclerose Lateral Amiotrófica/complicações , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas Relacionadas à Autofagia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Demência Frontotemporal/etiologia , Demência Frontotemporal/metabolismo , Humanos , Corpos de Inclusão/metabolismo , Camundongos , Neurônios Motores/metabolismo , UbiquitinaçãoRESUMO
Amyotrophic lateral sclerosis (ALS)-linked mutations in UBQLN2 and some members of the heterogeneous nuclear ribonucleoproteins (hnRNPs) family cause ALS. Most mutations in UBQLN2 are missense mutations that occur in and around a PXX repeat motif located in the central domain of the encoded protein. However, neither the function of the PXX motif nor the mechanism by which mutations in UBQLN2 cause ALS is known. We screened a yeast two-hybrid library using the central domain of ubiquilin-2 hoping to identify proteins whose binding is affected by the UBQLN2 mutations. Three such interactors were identified-hnRNPA1, hnRNPA3 and hnRNPU-all members of the hnRNP family. The interacting region in each of these proteins was their glycine-rich domain, the domain most frequently mutated in hnRNP-related proteins that cause ALS. We focused on hnRNPA1, because a mutation in the protein causes ALS. We confirmed the interaction between wild-type (WT) ubiquilin-2 and hnRNPA1 proteins in vitro and in cells. In contrast, all five ALS mutations in ubiquilin-2 that we examined had reduced binding with WT hnRNPA1. In addition, hnRNPA1 carrying the D262V missense mutation that causes ALS failed to bind WT ubiquilin-2. Overexpression of ubiquilin-2 containing the ALS mutations increased cell death and, for several of the mutants, this correlated with increased translocation of hnRNPA1 to the cytoplasm. Knockdown of ubiquilin-2 led to increased turnover of hnRNPA1, indicating ubiquilin-2 functions to stabilize hnRNPA1. The discovery that ubiquilin-2 interacts with hnRNP proteins and that mutation in either protein disrupts interaction suggests a connection between proteostasis and RNA metabolism.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ciclo Celular/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Mutação , Ubiquitinas/genética , Proteínas Adaptadoras de Transdução de Sinal , Alelos , Substituição de Aminoácidos , Esclerose Lateral Amiotrófica/metabolismo , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/metabolismo , Morte Celular/genética , Linhagem Celular , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Espaço Intracelular/metabolismo , Pressão Osmótica , Ligação Proteica , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinas/metabolismoRESUMO
Mutations in ATP13A2 (PARK9) have been linked to juvenile parkinsonism with dementia or Kufor-Rakeb syndrome (KRS). The ATP13A2 gene encodes at least three protein isoforms that arise by alternate splicing. A previous study indicated the Atp13a2(Isoform-1) protein is localized to lysosomes, whereas three separate mutations involved in disease cause retention of the protein in the ER. One speculation is that the mutant Atp13a2(Isoform-1) proteins are misfolded and eliminated by the ER-associated degradation pathway (ERAD), which involves the dislocation of proteins from the ER to the cytoplasm for proteasome degradation. We examined whether Atp13a2 proteins are degraded by ERAD and whether the Atp13a2(Isoform-3) protein has similar localization to the Atp13a2(Isoform-1) protein. Through analysis of protein turnover and by disrupting different steps in the ERAD pathway we demonstrate that mutant Atp13a2(Isoform-1) proteins are indeed eliminated by ERAD. Thus, siRNA-mediated knockdown of erasin, a platform for assembly of an ERAD complex, or expression of a dominant negative form of p97/VCP, a protein essential for dislocation of ERAD substrates, or inhibition of the proteasome all slowed degradation of the mutant Atp13a2(Isoform-1) proteins, but not the wild-type Atp13a2(Isoform-1) protein. Immunoprecipitation assays confirmed that the Atp13a2 proteins are ubiquitinated in accord with degradation by ERAD. In contrast to Atp13a2(Isoform-1), we show Atp13a2(Isoform-3) is localized to the ER and rapidly degraded. Lastly, we show Atp13a2 mutants have increased cytotoxicity and predispose cells to ER-stress-induced cell death. These results provide new insight into the properties of wild-type and mutant Atp13a2 proteins involved in KRS.
Assuntos
Estresse do Retículo Endoplasmático , Degradação Associada com o Retículo Endoplasmático , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/fisiopatologia , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Morte Celular , Linhagem Celular , Humanos , Mutação , Transtornos Parkinsonianos/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Here we present evidence, based on alterations of its intrinsic tryptophan fluorescence, that UBQLN2 protein undergoes a conformational switch when the temperature is raised from 37 °C to 42 °C. The switch is reset on restoration of the temperature. We speculate that the switch regulates UBQLN2 function in the heat shock response because elevation of the temperature from 37 °C to 42 °C dramatically increased in vitro binding between UBQLN2 and HSPA1B. Furthermore, restoration of the temperature to 37 °C decreased HSPA1B binding. By comparison to wild type (WT) UBQLN2, we found that all five ALS/FTD mutant UBQLN2 proteins we examined had attenuated alterations in tryptophan fluorescence when shifted to 42 °C, suggesting that the conformational switch is crippled in the mutants. Paradoxically, all five mutants bound similar amounts of HSPA1B compared to WT UBQLN2 protein at 42 °C, suggesting that either the conformational switch is not instrumental for HSPA1B binding, or that, although damaged, it is still functional. Comparison of the poly-ubiquitin chain binding revealed that WT UBQLN2 binds more avidly with K63 than with K48 chains. The avidity may explain the involvement of UBQLN2 in autophagy and cell signaling. Consistent with its function in autophagy, we found UBQLN2 binds directly with LC3, the autophagosomal-specific membrane-tethered protein. Finally, we provide evidence that WT UBQLN2 can homodimerize, and heterodimerize with WT UBQLN1. We show that ALS mutant P497S-UBQLN2 protein can oligomerize with either WT UBQLN1 or 2, providing a possible mechanism for how mutant UBQLN2 proteins could bind and inactivate UBQLN proteins, causing loss of function.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/genética , Temperatura , Triptofano/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Mutação , Proteínas de Choque Térmico HSP70/genéticaRESUMO
The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin ß, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin ß specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin ß is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin ß cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin ß in ER protein quality control.
Assuntos
Retículo Endoplasmático/metabolismo , Mutação , Ubiquitinação/fisiologia , Resposta a Proteínas não Dobradas/fisiologia , alfa 1-Antitripsina/metabolismo , beta Carioferinas/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/genética , Células HEK293 , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico/fisiologia , alfa 1-Antitripsina/genética , beta Carioferinas/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
Autophagy is the process by which organelles and portions of the cytoplasm are degraded in lysosomes. Several different forms of autophagy are known that are distinguishable chiefly by the mode in which cargo is delivered to the lysosome for degradation. Ubiquilin was recently reported to regulate macroautophagy, the form of autophagy in which cytosolic cargo is packaged in a double-membrane structure or autophagosome that fuses with lysosomes for degradation. We confirm here using different morphological and biochemical procedures that ubiquilin is present in autophagosomes in HeLa cells and in brain and liver tissue of mouse. Coimmunoprecipitation studies indicated that ubiquilin binds the autophagosome marker LC3 in a complex and that reduction of ubiquilin expression reduces autophagosome formation, which correlates with a reduction in maturation of LC3-I to the LC3-II form of the protein. We found that ubiquilin is degraded during both macroautophagy and during chaperone-mediated autophagy (CMA), the latter of which involves the active transport of proteins into lysosomes. We discuss the implication of this degradation in mediating cross-talk between macroautophagy and CMA. Finally, we demonstrate that ubiquilin protects cells against starvation-induced cell death propagated by overexpression of mutant Alzheimer's disease PS2N141I protein and green fluorescent protein (GFP)-huntingtin exon-1 fusion protein containing 74 polyglutamines.
Assuntos
Autofagia , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia , Western Blotting , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteína Huntingtina , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Peptídeos/genética , Fagossomos/metabolismo , Presenilina-2/genética , Presenilina-2/metabolismo , Ligação Proteica , Interferência de RNA , Transfecção , Repetições de Trinucleotídeos/genéticaRESUMO
C9ORF72-hexanucleotide repeat expansions and ubiquilin-2 (UBQLN2) mutations are recently identified genetic markers in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We investigate the relationship between C9ORF72 expansions and the clinical phenotype and neuropathology of ALS and FTLD. Genetic analysis and immunohistochemistry (IHC) were performed on autopsy-confirmed ALS (N = 75), FTLD-TDP (N = 30), AD (N = 14), and controls (N = 11). IHC for neurodegenerative disease pathology consisted of C9ORF72, UBQLN, p62, and TDP-43. A C9ORF72 expansion was identified in 19.4 % of ALS and 31 % of FTLD-TDP cases. ALS cases with C9ORF72 expansions frequently showed a bulbar onset of disease (57 %) and more rapid disease progression to death compared to non-expansion cases. Staining with C9ORF72 antibodies did not yield specific pathology. UBQLN pathology showed a highly distinct pattern in ALS and FTLD-TDP cases with the C9ORF72 expansion, with UBQLN-positive cytoplasmic inclusions in the cerebellar granular layer and extensive UBQLN-positive aggregates and dystrophic neurites in the hippocampal molecular layer and CA regions. These UBQLN pathologies were sufficiently unique to allow correct prediction of cases that were later confirmed to have C9ORF72 expansions by genetic analysis. UBQLN pathology partially co-localized with p62, and to a minor extent with TDP-43 positive dystrophic neurites and spinal cord skein-like inclusions. Our data indicate a pathophysiological link between C9ORF72 expansions and UBQLN proteins in ALS and FTLD-TDP that is associated with a highly characteristic pattern of UBQLN pathology. Our study indicates that this pathology is associated with alterations in clinical phenotype, and suggests that the presence of C9ORF72 repeat expansions may indicate a worse prognosis in ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ciclo Celular/metabolismo , Degeneração Lobar Frontotemporal/genética , Proteínas/metabolismo , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/mortalidade , Esclerose Lateral Amiotrófica/patologia , Proteínas Relacionadas à Autofagia , Proteína C9orf72 , Proteínas de Ciclo Celular/genética , Expansão das Repetições de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Degeneração Lobar Frontotemporal/mortalidade , Degeneração Lobar Frontotemporal/patologia , Humanos , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Proteínas/genética , Ubiquitinas/genéticaRESUMO
Ubiquilin (UBQLN) proteins are a dynamic and versatile family of proteins found in all eukaryotes that function in the regulation of proteostasis. Besides their canonical function as shuttle factors in delivering misfolded proteins to the proteasome and autophagy systems for degradation, there is emerging evidence that UBQLN proteins play broader roles in proteostasis. New information suggests the proteins function as chaperones in protein folding, protecting proteins prior to membrane insertion, and as guardians for mitochondrial protein import. In this review, we describe the evidence for these different roles, highlighting how different domains of the proteins impart these functions. We also describe how changes in the structure and phase separation properties of UBQLNs may regulate their activity and function. Finally, we discuss the pathogenic mechanisms by which mutations in UBQLN2 cause amyotrophic lateral sclerosis and frontotemporal dementia. We describe the animal model systems made for different UBQLN2 mutations and how lessons learnt from these systems provide fundamental insight into the molecular mechanisms by which UBQLN2 mutations drive disease pathogenesis through disturbances in proteostasis.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Demência Frontotemporal/genética , Proteínas Mitocondriais/genética , Mutação , Proteínas Nucleares/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMO
Huntington's disease (HD) is caused by an expansion of a CAG trinucleotide sequence that encodes a polyglutamine tract in the huntingtin (Htt) protein. Expansion of the polyglutamine tract above 35 repeats causes disease, with the age of onset inversely related to the degree of expansion above this number. Growing evidence suggests that mitochondrial function is compromised during HD pathogenesis, but how this occurs is not understood. We examined mitochondrial properties of HeLa cells that expressed green fluorescent protein (GFP)- or FLAG-tagged N-terminal portions of the Htt protein containing either, 17, 28, 74 or 138 polyglutamine repeats. Immunofluorescence staining of cells using antibodies against Tom20, a mitochondrion localized protein, revealed that cells expressing Htt proteins with 74 or 138 polyglutamine repeats were more sensitized to oxidative stress-induced mitochondria fragmentation and had reduced ATP levels compared with cells expressing Htt proteins with 17 or 28 polyglutamine repeats. By measuring changes in fluorescence of a photoactivated GFP protein targeted to mitochondria, we found that cells expressing red fluorescent protein (RFP)-tagged Htt protein containing 74 polyglutamine repeats had mitochondria that displayed reduced movement and fusion than cells expressing RFP-Htt protein with 28 polyglutamine repeats. Overexpression of Drp-1(K38A), a dominant-negative mitochondria-fission mutant, or Mfn2, a protein that promotes mitochondria fusion, suppressed polyglutamine-induced mitochondria fragmentation, the reduction of ATP levels and cell death. In a Caenorhabditis elegans model of HD, we found that reduction of Drp-1 expression by RNA interference rescued the motility defect associated with the expression of Htt proteins with polyglutamine repeats. These results suggest that the increase in cytotoxicity induced by Htt proteins containing expanded polyglutamine tracts is likely mediated, at least in part, by an alteration in normal mitochondrial dynamics, which results in increased mitochondrial fragmentation. Furthermore, our results suggest that it might be possible to reverse polyglutamine-induced cytotoxicity by preventing mitochondrial fragmentation.
Assuntos
Doença de Huntington/metabolismo , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Expansão das Repetições de Trinucleotídeos , Trifosfato de Adenosina/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Morte Celular , Fragmentação do DNA , Dinaminas , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica , Células HeLa , Humanos , Proteína Huntingtina , Doença de Huntington/genética , Doença de Huntington/fisiopatologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/química , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estresse Oxidativo , Peptídeos/genética , Peptídeos/metabolismoRESUMO
Accumulating evidence suggests X-linked dominant mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD) through both loss- and gain-of-function mechanisms. However, the mechanisms by which the mutations cause disease are still unclear. The goal of the study was to uncover the possible pathomechanism(s) by which UBQLN2 mutations cause ALS/FTD. An analysis of proteomic changes in neuronal tissue was used to identify proteins with altered accumulation in the P497S UBQLN2 transgenic mouse model of ALS/FTD. We then used immunocytochemistry and biochemical techniques to confirm protein changes in the mutant P497S mice. Additionally, we used cell lines inactivated of UBQLN2 expression to determine whether its loss underlies the alteration in the proteins seen in P497S mice. The proteome screen identified a dramatic alteration of serine protease inhibitor (serpin) proteins in the mutant P497S animals. Double immunofluorescent staining of brain and spinal cord tissues of the mutant and control mice revealed an age-dependent change in accumulation of Serpin A1, C1, and I1 in puncta whose staining colocalized with UBQLN2 puncta in the mutant P497S mice. Serpin A1 aggregation in P497S animals was confirmed by biochemical extraction and filter retardation assays. A similar phenomenon of serpin protein aggregation was found in HeLa and NSC34 motor neuron cells with inactivated UBQLN2 expression. We found aberrant aggregation of serpin proteins, particularly Serpin A1, in the brain and spinal cord of the P497S UBQLN2 mouse model of ALS/FTD. Similar aggregation of serpin proteins was found in UBQLN2 knockout cells suggesting that serpin aggregation in the mutant P497S animals may stem from loss of UBQLN2 function. Because serpin aggregation is known to cause disease through both loss- and gain-of-function mechanisms, we speculate that their accumulation in the P497S mouse model of ALS/FTD may contribute to disease pathogenesis through similar mechanism(s).
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas Relacionadas à Autofagia/metabolismo , Demência Frontotemporal/patologia , Serpinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Esclerose Lateral Amiotrófica/metabolismo , Animais , Proteínas Relacionadas à Autofagia/genética , Modelos Animais de Doenças , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Serpinas/metabolismo , Medula Espinal/patologiaRESUMO
Missense mutations in UBQLN2 cause X-linked dominant inheritance of amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). UBQLN2 belongs to a family of four highly homologous proteins expressed in humans that play diverse roles in maintaining proteostasis, but whether one isoform can substitute for another is not known. Here, we tested whether overexpression of UBQLN1 can alleviate disease in the P497S UBQLN2 mouse model of ALS/FTD by crossing transgenic (Tg) mouse lines expressing the two proteins and characterizing the resulting genotypes using a battery of pathologic and behavioral tests. The pathologic findings revealed UBQLN1 overexpression dramatically reduced the burden of UBQLN2 inclusions, neuronal loss and disturbances in proteostasis in double Tg mice compared to single P497S Tg mice. The beneficial effects of UBQLN1 overexpression were primarily confirmed by behavioral improvements seen in rotarod performance and grip strength in male, but not female mice. Paradoxically, although UBQLN1 overexpression reduced pathologic signatures of disease in P497S Tg mice, female mice had larger percentage of body weight loss than males, and this correlated with a corresponding lack of behavioral improvements in the females. These findings lead us to speculate that methods to upregulate UBQLN1 expression may reduce pathogenicity caused by UBQLN2 mutations, but may also lead to gender-specific outcomes that will have to be carefully weighed with the therapeutic benefits of UBQLN1 upregulation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Medula Espinal/patologiaRESUMO
UBQLN proteins regulate proteostasis by facilitating clearance of misfolded proteins through the proteasome and autophagy degradation pathways. Consistent with its proteasomal function, UBQLN proteins contain both UBL and UBA domains, which bind subunits of the proteasome, including the S5a subunit, and ubiquitin chains, respectively. Conclusions regarding the binding properties of UBQLN proteins have been derived principally through studies of its individual domains, not the full-length (FL) proteins. Here we describe the in vitro binding properties of FL-UBQLN1 with the S5a subunit of the proteasome and two different lysine-linked (K48 or K63) ubiquitin chains. We show that in contrast to its isolated UBA domain, which binds almost equally well with both K48 and K63 ubiquitin chains, FL UBQLN1 binds preferentially with K63 chains. Furthermore, we show that deletion of the UBL domain from UBQLN1 abrogates ubiquitin binding. Taken together these results suggest that sequences outside of the UBA domain in UBQLN1 function to regulate the specificity and binding with different ubiquitin moieties. We also show that the UBL domain of UBQLN1 is required for S5a binding and that its binding to UBQLN1, in turn, enhances K48 ubiquitin chain binding to the complex. We discuss the implications of our findings with the known function of UBQLN proteins in protein degradation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Ubiquitina/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Domínios Proteicos , ProteóliseRESUMO
In this issue of Structure, Dao et al. (2019) report that ALS-linked mutations in the Pxx domain of Ubiquilin 2 (UBQLN2) differentially influence the protein's phase separation abilities. The affect is by reducing the temperature and UBQLN2 concentration necessary for liquid-liquid phase separation droplet formation and by modulating UBQLN2 oligomerization.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas Adaptadoras de Transdução de Sinal , Aminoácidos , Proteínas Relacionadas à Autofagia , Proteínas de Ciclo Celular/genética , Humanos , Mutação , Ubiquitinas/genéticaRESUMO
Mutations in ATP13A2 cause Kufor-Rakeb syndrome (KRS), a juvenile form of Parkinson's disease (PD) with dementia. However, the mechanisms by which mutations in ATP13A2 cause KRS is not understood. The mutations lead to misfolding of the translated Atp13a2 protein and its premature degradation in the endoplasmic reticulum, never reaching the lysosome where the protein is thought to function. Atp13a2 is a P-type ATPase, a class of proteins that function in ion transport. Indeed, studies of human, mouse, and yeast Atp13a2 proteins suggest a possible involvement in regulation of heavy metal toxicity. Here we report on the cytoprotective function of Atp13a2 on HeLa cells and dopamine neurons of Caenorhabditis elegans (C. elegans). HeLa cells stably overexpressing V5- tagged Atp13a2Isoform-1 protein were more resistant to elevated manganese exposure and to starvation-induced cell death compared to cells not overexpressing the protein. Because PD is characterized by loss of dopamine neurons, we generated transgenic C. elegans expressing GFP-tagged human Atp13a2 protein in dopamine neurons. The transgenic animals exhibited higher resistance to dopamine neuron degeneration after acute exposure to manganese compared to nematodes that expressed GFP alone. The results suggest Atp13a2 Isoform-1 protein confers cytoprotection against toxic insults, including those that cause PD syndromes.
Assuntos
Morte Celular , Manganês/toxicidade , ATPases Translocadoras de Prótons/farmacologia , Inanição , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Neurônios Dopaminérgicos/efeitos dos fármacos , Células HeLa , Humanos , Doença de Parkinson/prevenção & controle , Substâncias Protetoras/metabolismo , Isoformas de Proteínas , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismoRESUMO
Pathogens exploit host machinery to establish an environment that favors their propagation. Because of their pivotal roles in cellular physiology, protein degradation pathways are common targets for viral proteins. Protein-linking integrin-associated protein and cytoskeleton 1 (PLIC1), also called ubiquilin, contains an amino-terminal ubiquitin-like (UBL) domain and a carboxy-terminal ubiquitin-associated (UBA) domain. PLIC1 is proposed to function as a regulator of the ubiquitination complex and proteasome machinery. Kaposi's sarcoma-associated herpesvirus (KSHV) contains a small membrane protein, K7, that protects cells from apoptosis induced by various stimuli. We report here that cellular PLIC1 is a K7-interacting protein and that the central hydrophobic region of K7 and the carboxy-terminal UBA domain of PLIC1 are responsible for their interaction. Cellular PLIC1 formed a dimer and bound efficiently to polyubiquitinated proteins through its carboxy-terminal UBA domain, and this activity correlated with its ability to stabilize cellular I kappa B protein. In contrast, K7 interaction prevented PLIC1 from forming a dimer and binding to polyubiquitinated proteins, leading to the rapid degradation of I kappa B. Furthermore, K7 expression promoted efficient degradation of the p53 tumor suppressor, resulting in inhibition of p53-mediated apoptosis. These results indicate that KSHV K7 targets a regulator of the ubiquitin- and proteasome-mediated degradation machinery to deregulate cellular protein turnover, which potentially provides a favorable environment for viral reproduction.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Herpesvirus Humano 8/metabolismo , Proteínas Mitocondriais/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Relacionadas à Autofagia , Proteínas de Transporte/genética , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Dimerização , Herpesvirus Humano 8/genética , Humanos , Proteínas I-kappa B/metabolismo , Proteínas Mitocondriais/genética , Complexos Multienzimáticos/metabolismo , Inibidor de NF-kappaB alfa , Complexo de Endopeptidases do Proteassoma , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitinas/genética , Proteínas Virais/genéticaRESUMO
Ubiquilin proteins have been shown to interact with a wide variety of other cellular proteins, often regulating the stability and degradation of the interacting protein. Ubiquilin contains a UBL (ubiquitin-like) domain at the N-terminus and a UBA (ubiquitin-associated) domain at the C-terminus, separated by a central region containing Sti1-like repeats. Little is known about regulation of the interaction of ubiquilin with other proteins. In the present study, we show that ubiquilin is capable of forming dimers, and that dimerization requires the central region of ubiquilin, but not its UBL or the UBA domains. Furthermore, we provide evidence suggesting that monomeric ubiquilin is likely to be the active form that is involved in binding presenilin proteins. Our results provide new insight into the regulatory mechanism underlying the interaction of ubiquilin with presenilins.