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1.
Hum Mol Genet ; 32(14): 2386-2398, 2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37220877

RESUMO

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 , Mutação
2.
Neurobiol Dis ; 192: 106421, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38286389

RESUMO

Previously, we demonstrated that the SCFcyclin F complex directly mediates the poly-ubiquitylation of TDP-43, raising the question of whether cyclin F can be used to enhance the turnover of TDP-43. A hurdle to the use of cyclin F, however, is that the overexpression of cyclin F can lead to the initiation of cell death pathways. Accordingly, the aim of this study was to identify and evaluate a less toxic variant of cyclin F. To do so, we first confirmed and validated our previous findings that cyclin F binds to TDP-43 in an atypical manner. Additionally, we demonstrated that mutating the canonical substrate region in cyclin F (to generate cyclin FMRL/AAA) led to reduced binding affinity to known canonical substrates without impacting the interaction between cyclin F and TDP-43. Notably, both wild-type and cyclin FMRL/AAA effectively reduced the abundance of TDP-43 in cultured cells whilst cyclin FMRL/AAA also demonstrated reduced cell death compared to the wild-type control. The decrease in toxicity also led to a reduction in morphological defects in zebrafish embryos. These results suggest that cyclin F can be modified to enhance its targeting of TDP-43, which in turn reduces the toxicity associated with the overexpression of cyclin F. This study provides greater insights into the interaction that occurs between cyclin F and TDP-43 in cells and in vivo.


Assuntos
Esclerose Lateral Amiotrófica , Animais , Esclerose Lateral Amiotrófica/metabolismo , Peixe-Zebra , Proteínas de Ligação a DNA/metabolismo , Ubiquitinação , Ciclinas/genética , Ciclinas/metabolismo
3.
J Neurochem ; 165(4): 563-586, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36847488

RESUMO

Progressive supranuclear palsy (PSP) is a late-onset neurodegenerative disease defined pathologically by the presence of insoluble phosphorylated-Tau (p-Tau) in neurons and glia. Identifying co-aggregating proteins within p-Tau inclusions may reveal important insights into processes affected by the aggregation of Tau. We used a proteomic approach, which combines antibody-mediated biotinylation and mass spectrometry (MS) to identify proteins proximal to p-Tau in PSP. Using this proof-of-concept workflow for identifying interacting proteins of interest, we characterized proteins proximal to p-Tau in PSP cases, identifying >84% of previously identified interaction partners of Tau and known modifiers of Tau aggregation, while 19 novel proteins not previously found associated with Tau were identified. Furthermore, our data also identified confidently assigned phosphorylation sites that have been previously reported on p-Tau. Additionally, using ingenuity pathway analysis (IPA) and human RNA-seq datasets, we identified proteins previously associated with neurological disorders and pathways involved in protein degradation, stress responses, cytoskeletal dynamics, metabolism, and neurotransmission. Together, our study demonstrates the utility of biotinylation by antibody recognition (BAR) approach to answer a fundamental question to rapidly identify proteins in proximity to p-Tau from post-mortem tissue. The application of this workflow opens up the opportunity to identify novel protein targets to give us insight into the biological process at the onset and progression of tauopathies.


Assuntos
Doenças Neurodegenerativas , Paralisia Supranuclear Progressiva , Tauopatias , Humanos , Proteínas tau/metabolismo , Paralisia Supranuclear Progressiva/metabolismo , Proteólise , Proteômica , Tauopatias/metabolismo , Transmissão Sináptica
4.
Hum Mol Genet ; 30(11): 971-984, 2021 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-33729478

RESUMO

Previously, we identified missense mutations in CCNF that are causative of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Hallmark features of these diseases include the build-up of insoluble protein aggregates as well as the mislocalization of proteins such as transactive response DNA binding protein 43 kDa (TDP-43). In recent years, the dysregulation of SFPQ (splicing factor proline and glutamine rich) has also emerged as a pathological hallmark of ALS/FTD. CCNF encodes for the protein cyclin F, a substrate recognition component of an E3 ubiquitin ligase. We have previously shown that ALS/FTD-linked mutations in CCNF cause disruptions to overall protein homeostasis that leads to a build-up of K48-linked ubiquitylated proteins as well as defects in autophagic machinery. To investigate further processes that may be affected by cyclin F, we used a protein-proximity ligation method, known as Biotin Identification (BioID), standard immunoprecipitations and mass spectrometry to identify novel interaction partners of cyclin F and infer further process that may be affected by the ALS/FTD-causing mutation. Results demonstrate that cyclin F closely associates with proteins involved with RNA metabolism as well as a number of RNA-binding proteins previously linked to ALS/FTD, including SFPQ. Notably, the overexpression of cyclin F(S621G) led to the aggregation and altered subcellular distribution of SFPQ in human embryonic kidney (HEK293) cells, while leading to altered degradation in primary neurons. Overall, our data links ALS/FTD-causing mutations in CCNF to converging pathological features of ALS/FTD and provides a link between defective protein degradation systems and the pathological accumulation of a protein involved in RNA processing and metabolism.


Assuntos
Esclerose Lateral Amiotrófica/genética , Ciclinas/genética , Demência Frontotemporal/genética , Fator de Processamento Associado a PTB/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteínas de Ligação a DNA/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Células HEK293 , Humanos , Agregados Proteicos/genética , Mapas de Interação de Proteínas/genética , Proteólise , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/genética
5.
Int J Mol Sci ; 24(5)2023 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-36901711

RESUMO

Proteomics offers vast potential for studying the molecular regulation of the human brain. Formalin fixation is a common method for preserving human tissue; however, it presents challenges for proteomic analysis. In this study, we compared the efficiency of two different protein-extraction buffers on three post-mortem, formalin-fixed human brains. Equal amounts of extracted proteins were subjected to in-gel tryptic digestion and LC-MS/MS. Protein, peptide sequence, and peptide group identifications; protein abundance; and gene ontology pathways were analyzed. Protein extraction was superior using lysis buffer containing tris(hydroxymethyl)aminomethane hydrochloride, sodium dodecyl sulfate, sodium deoxycholate, and Triton X-100 (TrisHCl, SDS, SDC, Triton X-100), which was then used for inter-regional analysis. Pre-frontal, motor, temporal, and occipital cortex tissues were analyzed by label free quantification (LFQ) proteomics, Ingenuity Pathway Analysis and PANTHERdb. Inter-regional analysis revealed differential enrichment of proteins. We found similarly activated cellular signaling pathways in different brain regions, suggesting commonalities in the molecular regulation of neuroanatomically-linked brain functions. Overall, we developed an optimized, robust, and efficient method for protein extraction from formalin-fixed human brain tissue for in-depth LFQ proteomics. We also demonstrate herein that this method is suitable for rapid and routine analysis to uncover molecular signaling pathways in the human brain.


Assuntos
Formaldeído , Proteômica , Humanos , Formaldeído/química , Cromatografia Líquida/métodos , Proteômica/métodos , Octoxinol , Espectrometria de Massas em Tandem/métodos , Proteínas/análise , Peptídeos , Encéfalo , Inclusão em Parafina , Fixação de Tecidos/métodos
6.
Neurobiol Dis ; 167: 105673, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35231559

RESUMO

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the loss of upper and lower motor neurons in the brain and spinal cord. ALS and frontotemporal dementia (FTD) are overlapping diseases with shared pathological features. Affected neurons of people with ALS and FTD typically contain ubiquitin-immunoreactive inclusions, of which TDP-43 (Tar DNA-binding protein of 43 kDa) is a major component. However, what triggers the formation of these abnormal TDP-43 inclusions is unclear. Previously, we identified CCNF mutations in cohorts of familial and sporadic cases of ALS and FTD. CCNF encodes cyclin F, the substrate-binding component of a multiprotein E3 ubiquitin ligase complex that ubiquitylates and subsequently directs a set of protein substrates for proteasomal degradation. Here, we explored the relationship between cyclin F and TDP-43. METHODS: We used a series of complementary biochemical approaches including immunoprecipitations, in vitro ubiquitylation assays, immunofluorescence imaging and immunocytochemistry. Unpaired student t-tests were used to determine statistical significance of the results. RESULTS: In this study, we demonstrate that that the SCFcyclin F complex directly mediates the poly-ubiquitylation of TDP-43. Importantly, we demonstrate that cyclin F bearing the pathogenic ALS/FTD mutation, S621G, leads to aberrant ubiquitylation of TDP-43 as well as the accumulation of K48-ubiquitylated TDP-43 in neuron-like cells. Furthermore, we demonstrate that a patient carrying the ALS/FTD cyclin FS195R mutation displayed skein-like cytoplasmic TDP-43 aggregates, implying abnormal TDP-43 degradation in a CCNF mutation bearing patient. CONCLUSION: In summary, this study reports a direct ubiquitylation mechanism for TDP-43, revealing important insights into the regulation of cyclin F-mediated TDP-43 turnover and clues towards understanding the molecular origins of the ubiquitylated TDP-43 inclusions that are the hallmark pathological feature in ALS and FTD.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Doenças Neurodegenerativas , Esclerose Lateral Amiotrófica/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Neurônios Motores/patologia , Doenças Neurodegenerativas/patologia , Ubiquitinação
7.
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
8.
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 Lateral Amiotrófica/genética , Ciclinas/genética , Demência Frontotemporal/genética , Medula Espinal/patologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/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
9.
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 Lateral Amiotrófica/genética , Autofagia/genética , Ciclinas/genética , Demência Frontotemporal/genética , Ubiquitinação/genética , Esclerose Lateral Amiotrófica/complicações , Células Cultivadas , Demência Frontotemporal/complicações , Células HEK293 , Humanos , Lisina/metabolismo , Mutação de Sentido Incorreto/fisiologia
11.
Ageing Res Rev ; 96: 102246, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38401571

RESUMO

TAR DNA binding protein-43 (TDP-43) is a key component in RNA splicing which plays a crucial role in the aging process. In neurodegenerative diseases such as amyotrophic lateral sclerosis, frontotemporal dementia and limbic-predominant age-related TDP-43 encephalopathy, TDP-43 can be mutated, mislocalised out of the nucleus of neurons and glial cells and form cytoplasmic inclusions. These TDP-43 alterations can lead to its RNA splicing dysregulation and contribute to mis-splicing of various types of RNA, such as mRNA, microRNA, and circular RNA. These changes can result in the generation of an altered transcriptome and proteome within cells, ultimately changing the diversity and quantity of gene products. In this review, we summarise the findings of novel atypical RNAs resulting from TDP-43 dysfunction and their potential as biomarkers or targets for therapeutic development.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Splicing de RNA , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neurônios/metabolismo
12.
Ageing Res Rev ; 92: 102085, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37813308

RESUMO

A common feature of adult-onset neurodegenerative diseases is the presence of characteristic pathological accumulations of specific proteins. These pathological protein depositions can vary in their protein composition, cell-type distribution, and intracellular (or extracellular) location. For example, abnormal cytoplasmic protein deposits which consist of the TDP-43 protein are found within motor neurons in patients with amyotrophic lateral sclerosis (ALS, a common form of motor neuron disease) and frontotemporal dementia (FTD). The presence of these insoluble intracellular TDP-43 inclusions suggests that restoring TDP-43 homeostasis represents a potential therapeutical strategy, which has been demonstrated in alleviating neurodegenerative symptoms in cell and animal models of ALS/FTD. We have reviewed the mechanisms that lead to disrupted TDP-43 homeostasis and discussed how small molecule-based therapies could be applied in modulating these mechanisms. This review covers recent advancements and challenges in small molecule-based therapies that could be used to clear pathological forms of TDP-43 through various protein homeostasis mechanisms and advance the way towards finding effective therapeutical drug discoveries for neurodegenerative diseases characterized by TDP-43 proteinopathies, especially ALS and FTD. We also consider the wider insight of these therapeutic strategies for other neurodegenerative diseases.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Doença dos Neurônios Motores , Doenças Neurodegenerativas , Animais , Humanos , Esclerose Lateral Amiotrófica/terapia , Proteínas de Ligação a DNA/metabolismo , Demência Frontotemporal/terapia , Doença dos Neurônios Motores/terapia , Doença dos Neurônios Motores/patologia , Doenças Neurodegenerativas/terapia
13.
Sci Rep ; 13(1): 20467, 2023 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-37993492

RESUMO

Amyotrophic lateral sclerosis (ALS) is a severely debilitating neurodegenerative condition that is part of the same disease spectrum as frontotemporal dementia (FTD). Mutations in the CCNF gene, encoding cyclin F, are present in both sporadic and familial ALS and FTD. However, the pathophysiological mechanisms underlying neurodegeneration remain unclear. Proper functioning of the endoplasmic reticulum (ER) and Golgi apparatus compartments is essential for normal physiological activities and to maintain cellular viability. Here, we demonstrate that ALS/FTD-associated variant cyclin FS621G inhibits secretory protein transport from the ER to Golgi apparatus, by a mechanism involving dysregulation of COPII vesicles at ER exit sites. Consistent with this finding, cyclin FS621G also induces fragmentation of the Golgi apparatus and activates ER stress, ER-associated degradation, and apoptosis. Induction of Golgi fragmentation and ER stress were confirmed with a second ALS/FTD variant cyclin FS195R, and in cortical primary neurons. Hence, this study provides novel insights into pathogenic mechanisms associated with ALS/FTD-variant cyclin F, involving perturbations to both secretory protein trafficking and ER-Golgi homeostasis.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Mutação , Ciclinas/metabolismo
14.
Mol Neurobiol ; 60(9): 5034-5054, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37243816

RESUMO

Amyotrophic lateral sclerosis (ALS)- and frontotemporal dementia (FTD)-linked mutations in CCNF have been shown to cause dysregulation to protein homeostasis. CCNF encodes for cyclin F, which is part of the cyclin F-E3 ligase complex SCFcyclinF known to ubiquitylate substrates for proteasomal degradation. In this study, we identified a function of cyclin F to regulate substrate solubility and show how cyclin F mechanistically underlies ALS and FTD disease pathogenesis. We demonstrated that ALS and FTD-associated protein sequestosome-1/p62 (p62) was a canonical substrate of cyclin F which was ubiquitylated by the SCFcyclinF complex. We found that SCFcyclin F ubiquitylated p62 at lysine(K)281, and that K281 regulated the propensity of p62 to aggregate. Further, cyclin F expression promoted the aggregation of p62 into the insoluble fraction, which corresponded to an increased number of p62 foci. Notably, ALS and FTD-linked mutant cyclin F p.S621G aberrantly ubiquitylated p62, dysregulated p62 solubility in neuronal-like cells, patient-derived fibroblasts and induced pluripotent stem cells and dysregulated p62 foci formation. Consistently, motor neurons from patient spinal cord tissue exhibited increased p62 ubiquitylation. We suggest that the p.S621G mutation impairs the functions of cyclin F to promote p62 foci formation and shift p62 into the insoluble fraction, which may be associated to aberrant mutant cyclin F-mediated ubiquitylation of p62. Given that p62 dysregulation is common across the ALS and FTD spectrum, our study provides insights into p62 regulation and demonstrates that ALS and FTD-linked cyclin F mutant p.S621G can drive p62 pathogenesis associated with ALS and FTD.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Esclerose Lateral Amiotrófica/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclinas/metabolismo , Ubiquitinação , Mutação/genética
15.
Neuroscientist ; : 10738584221120182, 2022 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-36062310

RESUMO

Amyotrophic lateral sclerosis (ALS) is the most common form of motor neuron disease and is characterized by the degeneration of upper and lower motor neurons of the brain and spinal cord. ALS is also linked clinically, genetically, and pathologically to a form of dementia known as frontotemporal dementia (FTD). Identifying gene mutations that cause ALS/FTD has provided valuable insight into the disease process. Several ALS/FTD-causing mutations occur within proteins with roles in protein clearance systems. This includes ALS/FTD mutations in CCNF, which encodes the protein cyclin F: a component of a multiprotein E3 ubiquitin ligase that mediates the ubiquitylation of substrates for their timely degradation. In this review, we provide an update on the link between ALS/FTD CCNF mutations and neurodegeneration.

16.
Front Mol Neurosci ; 14: 627740, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33986643

RESUMO

The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a diverse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1/p62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an example, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of in vitro and in vivo models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein-protein and protein-substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies.

17.
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.

18.
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
19.
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
20.
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 Lateral Amiotrófica/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
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