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1.
Front Neurosci ; 13: 548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31244593

RESUMO

Neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are increasing in prevalence but lack targeted therapeutics. Although the pathological mechanisms behind these diseases remain unclear, both ALS and FTD are characterized pathologically by aberrant protein aggregation and inclusion formation within neurons, which correlates with neurodegeneration. Notably, aggregation of several key proteins, including TAR DNA binding protein of 43 kDa (TDP-43), superoxide dismutase 1 (SOD1), and tau, have been implicated in these diseases. Proteomics methods are being increasingly applied to better understand disease-related mechanisms and to identify biomarkers of disease, using model systems as well as human samples. Proteomics-based approaches offer unbiased, high-throughput, and quantitative results with numerous applications for investigating proteins of interest. Here, we review recent advances in the understanding of ALS and FTD pathophysiology obtained using proteomics approaches, and we assess technical and experimental limitations. We compare findings from various mass spectrometry (MS) approaches including quantitative proteomics methods such as stable isotope labeling by amino acids in cell culture (SILAC) and tandem mass tagging (TMT) to approaches such as label-free quantitation (LFQ) and sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) in studies of ALS and FTD. Similarly, we describe disease-related protein-protein interaction (PPI) studies using approaches including immunoprecipitation mass spectrometry (IP-MS) and proximity-dependent biotin identification (BioID) and discuss future application of new techniques including proximity-dependent ascorbic acid peroxidase labeling (APEX), and biotinylation by antibody recognition (BAR). Furthermore, we explore the use of MS to detect post-translational modifications (PTMs), such as ubiquitination and phosphorylation, of disease-relevant proteins in ALS and FTD. We also discuss upstream technologies that enable enrichment of proteins of interest, highlighting the contributions of new techniques to isolate disease-relevant protein inclusions including flow cytometric analysis of inclusions and trafficking (FloIT). These recently developed approaches, as well as related advances yet to be applied to studies of these neurodegenerative diseases, offer numerous opportunities for discovery of potential therapeutic targets and biomarkers for ALS and FTD.

2.
Cell Mol Life Sci ; 76(13): 2499-2510, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30919022

RESUMO

Ubiquitin ligases play an integral role in fine-tuning signaling cascades necessary for normal cell function. Aberrant regulation of ubiquitin ligases has been implicated in several neurodegenerative diseases, generally, due to mutations within the E3 ligase itself. Several proteomic-based methods have recently emerged to facilitate the rapid identification of ligase-substrate pairs-a previously challenging feat due to the transient nature of ligase-substrate interactions. These novel methods complement standard immunoprecipitations (IPs) and include proximity-dependent biotin identification (BioID), ubiquitin ligase-substrate trapping, tandem ubiquitin-binding entities (TUBEs), and a molecular trapping unit known as the NEDDylator. The implementation of these techniques is expected to facilitate the rapid identification of novel substrates of E3 ubiquitin ligases, a process that is likely to enhance our understanding of neurodegenerative diseases and highlight novel therapeutic targets for the treatment of neurodegenerative diseases.


Assuntos
Terapia de Alvo Molecular , Doenças Neurodegenerativas/metabolismo , Proteômica/métodos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Especificidade por Substrato , Ubiquitinação
4.
Cell Mol Life Sci ; 75(2): 335-354, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28852778

RESUMO

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/genética , Autofagia/genética , Ciclinas/genética , Demência Frontotemporal/genética , Ubiquitinação/genética , Esclerose Amiotrófica Lateral/complicações , Células Cultivadas , Demência Frontotemporal/complicações , Células HEK293 , Humanos , Lisina/metabolismo , Mutação de Sentido Incorreto/fisiologia
5.
Open Biol ; 7(10)2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-29021214

RESUMO

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 , Ubiquitinação
6.
Int J Biochem Cell Biol ; 89: 216-220, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28652210

RESUMO

Cyclin F, encoded by CCNF, is the substrate recognition component of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCFcyclin F. E3 ubiquitin ligases play a key role in ubiquitin-proteasome mediated protein degradation, an essential component of protein homeostatic mechanisms within the cell. By recognising and regulating the availability of several protein substrates, SCFcyclin F plays a role in regulating various cellular processes including replication and repair of DNA and cell cycle checkpoint control. Cyclin F dysfunction has been implicated in various forms of cancer and CCNF mutations were recently linked to familial and sporadic amyotrophic lateral sclerosis and frontotemporal dementia, offering a new lead to understanding the pathogenic mechanisms underlying neurodegeneration. In this review, we evaluate the current literature on the function of cyclin F with an emphasis on its roles in cancer and neurodegeneration.


Assuntos
Ciclinas/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Ciclinas/química , Ciclinas/genética , Regulação da Expressão Gênica , Humanos , Neoplasias/patologia , Doenças Neurodegenerativas/patologia
7.
Hum Mol Genet ; 26(14): 2616-2626, 2017 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-28444311

RESUMO

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations.


Assuntos
Esclerose Amiotrófica Lateral/genética , Ciclinas/genética , Demência Frontotemporal/genética , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Animais , Animais Geneticamente Modificados , Axônios/patologia , Caspase 3/metabolismo , Morte Celular/genética , Ciclinas/biossíntese , Ciclinas/metabolismo , Modelos Animais de Doenças , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Mutação de Sentido Incorreto , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Peixe-Zebra
8.
Nat Commun ; 7: 11253, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-27080313

RESUMO

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase complex (SCF(Cyclin F)). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCF(Cyclin F) substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.


Assuntos
Esclerose Amiotrófica Lateral/genética , Ciclinas/genética , Demência Frontotemporal/genética , Predisposição Genética para Doença/genética , Mutação de Sentido Incorreto , Adulto , Idoso , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Mapeamento Cromossômico , Cromossomos Humanos Par 16/genética , Saúde da Família , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de DNA/métodos , Homologia de Sequência de Aminoácidos
9.
Front Cell Neurosci ; 9: 414, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26578880

RESUMO

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two progressive, fatal neurodegenerative syndromes with considerable clinical, genetic and pathological overlap. Clinical symptoms of FTD can be seen in ALS patients and vice versa. Recent genetic discoveries conclusively link the two diseases, and several common molecular players have been identified (TDP-43, FUS, C9ORF72). The definitive etiologies of ALS and FTD are currently unknown and both disorders lack a cure. Glia, specifically astrocytes and microglia are heavily implicated in the onset and progression of neurodegeneration witnessed in ALS and FTD. In this review, we summarize the current understanding of the role of microglia and astrocytes involved in ALS and FTD, highlighting their recent implications in neuroinflammation, alterations in waste clearance involving phagocytosis and the newly described glymphatic system, and vascular abnormalities. Elucidating the precise mechanisms of how astrocytes and microglia are involved in ALS and FTD will be crucial in characterizing these two disorders and may represent more effective interventions for disease progression and treatment options in the future.

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