RESUMO
Machado-Joseph disease (MJD) is an autosomal dominant neurodegenerative spinocerebellar ataxia caused by a polyglutamine-coding CAG repeat expansion in the ATXN3 gene. While the CAG length correlates negatively with the age at onset, it accounts for approximately 50% of its variability only. Despite larger efforts in identifying contributing genetic factors, candidate genes with a robust and plausible impact on the molecular pathogenesis of MJD are scarce. Therefore, we analysed missense single nucleotide polymorphism variants in the PRKN gene encoding the Parkinson's disease-associated E3 ubiquitin ligase parkin, which is a well-described interaction partner of the MJD protein ataxin-3, a deubiquitinase. By performing a correlation analysis in the to-date largest MJD cohort of more than 900 individuals, we identified the V380L variant as a relevant factor, decreasing the age at onset by 3 years in homozygous carriers. Functional analysis in an MJD cell model demonstrated that parkin V380L did not modulate soluble or aggregate levels of ataxin-3 but reduced the interaction of the two proteins. Moreover, the presence of parkin V380L interfered with the execution of mitophagy-the autophagic removal of surplus or damaged mitochondria-thereby compromising cell viability. In summary, we identified the V380L variant in parkin as a genetic modifier of MJD, with negative repercussions on its molecular pathogenesis and disease age at onset.
Assuntos
Doença de Machado-Joseph , Mitofagia , Ubiquitina-Proteína Ligases , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/patologia , Humanos , Ubiquitina-Proteína Ligases/genética , Mitofagia/genética , Mitofagia/fisiologia , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Polimorfismo de Nucleotídeo Único , Ataxina-3/genética , Idade de Início , Proteínas RepressorasRESUMO
Lysine residues are one of the main sites for posttranslational modifications of proteins, and lysine ubiquitination of the Machado-Joseph disease protein ataxin-3 is implicated in its cellular function and polyglutamine expansion-dependent toxicity. Despite previously undertaken efforts, the individual roles of specific lysine residues of the ataxin-3 sequence are not entirely understood and demand further analysis. By retaining single lysine residues of otherwise lysine-free wild-type and polyglutamine-expanded ataxin-3, we assessed the effects of a site-limited modifiability on ataxin-3 protein levels, aggregation propensity, localization, and stability. We confirmed earlier findings that levels of lysine-free ataxin-3 are reduced due to its decreased stability, which led to a diminished load of SDS-insoluble species of its polyglutamine-expanded form. The isolated presence of several single lysine residues within the N-terminus of polyglutamine-expanded ataxin-3 significantly restored its aggregate levels, with highest fold changes induced by the presence of lysine 8 or lysine 85, respectively. Ataxin-3 lacking all lysine residues presented a slightly increased nuclear localization, which was counteracted by the reintroduction of lysine 85, whereas presence of either lysine 8 or lysine 85 led to a significantly higher ataxin-3 stability. Moreover, lysine-free ataxin-3 showed increased toxicity and binding to K48-linked polyubiquitin chains, whereas the reintroduction of lysine 85, located between the ubiquitin-binding sites 1 and 2 of ataxin-3, normalized its binding affinity. Overall, our data highlight the relevance of lysine residues 8 and 85 of ataxin-3 and encourage further analyses, to evaluate the potential of modulating posttranslational modifications of these sites for influencing pathophysiological characteristics of the Machado-Joseph disease protein.
RESUMO
Retinoblastoma is a rare childhood tumor caused by the inactivation of both copies of the RB1 gene. Early diagnosis and identification of heritable RB1 mutation carriers can improve the disease outcome and management via genetic counseling. We used the Multiplex Ligation-dependent Probe Amplification (MLPA) method to analyze the RB1 gene and flanking regions in blood samples from 159 retinoblastoma patients previously negative for RB1 point mutations via Sanger sequencing. We detected a wide spectrum of germline chromosomal alterations, ranging from partial loss or duplication of RB1 to large deletions spanning RB1 and adjacent genes. Mutations were validated via karyotyping, fluorescent in situ hybridization (FISH), SNP-arrays (Single Nucleotide Polymorphism-arrays) and/or quantitative relative real-time PCR. Patients with leukocoria as a presenting symptom showed reduced death rate (p = 0.013) and this sign occurred more frequently among carriers of two breakpoints within RB1 (p = 0.05). All unilateral cases presented both breakpoints outside of RB1 (p = 0.0075). Patients with one breakpoint within RB1 were diagnosed at earlier ages (p = 0.017). Our findings characterize the mutational spectrum of a Brazilian cohort of retinoblastoma patients and point to a possible relationship between the mutation breakpoint location and tumor outcome, contributing to a better prospect of the genotype/phenotype correlation and adding to the wide diversity of germline mutations involving RB1 and adjacent regions in retinoblastoma.
Assuntos
Neoplasias da Retina , Retinoblastoma , Humanos , Retinoblastoma/diagnóstico , Retinoblastoma/genética , Retinoblastoma/patologia , Hibridização in Situ Fluorescente , Brasil/epidemiologia , Genes do Retinoblastoma/genética , Mutação , Neoplasias da Retina/patologia , Análise Mutacional de DNARESUMO
Machado-Joseph disease (MJD) is characterized by a pathological expansion of the polyglutamine (polyQ) tract within the ataxin-3 protein. Despite its primarily cytoplasmic localization, polyQ-expanded ataxin-3 accumulates in the nucleus and forms intranuclear aggregates in the affected neurons. Due to these histopathological hallmarks, the nucleocytoplasmic transport machinery has garnered attention as an important disease relevant mechanism. Here, we report on MJD cell model-based analysis of the nuclear transport receptor karyopherin subunit beta-1 (KPNB1) and its implications in the molecular pathogenesis of MJD. Although directly interacting with both wild-type and polyQ-expanded ataxin-3, modulating KPNB1 did not alter the intracellular localization of ataxin-3. Instead, overexpression of KPNB1 reduced ataxin-3 protein levels and the aggregate load, thereby improving cell viability. On the other hand, its knockdown and inhibition resulted in the accumulation of soluble and insoluble ataxin-3. Interestingly, the reduction of ataxin-3 was apparently based on protein fragmentation independent of the classical MJD-associated proteolytic pathways. Label-free quantitative proteomics and knockdown experiments identified mitochondrial protease CLPP as a potential mediator of the ataxin-3-degrading effect induced by KPNB1. We confirmed reduction of KPNB1 protein levels in MJD by analyzing two MJD transgenic mouse models and induced pluripotent stem cells (iPSCs) derived from MJD patients. Our results reveal a yet undescribed regulatory function of KPNB1 in controlling the turnover of ataxin-3, thereby highlighting a new potential target of therapeutic value for MJD.
Assuntos
Ataxina-3 , Endopeptidase Clp , Doença de Machado-Joseph , Mitocôndrias , beta Carioferinas , Animais , Ataxina-3/genética , Ataxina-3/metabolismo , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Camundongos , Mitocôndrias/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismoRESUMO
Spinocerebellar ataxia type 17 (SCA17) is a neurodegenerative disease caused by a polyglutamine-encoding trinucleotide repeat expansion in the gene of transcription factor TATA box-binding protein (TBP). While its underlying pathomechanism is elusive, polyglutamine-expanded TBP fragments of unknown origin mediate the mutant protein's toxicity. Calcium-dependent calpain proteases are protagonists in neurodegenerative disorders. Here, we demonstrate that calpains cleave TBP, and emerging C-terminal fragments mislocalize to the cytoplasm. SCA17 cell and rat models exhibited calpain overactivation, leading to excessive fragmentation and depletion of neuronal proteins in vivo. Transcriptome analysis of SCA17 cells revealed synaptogenesis and calcium signaling perturbations, indicating the potential cause of elevated calpain activity. Pharmacological or genetic calpain inhibition reduced TBP cleavage and aggregation, consequently improving cell viability. Our work underlines the general significance of calpains and their activating pathways in neurodegenerative disorders and presents these proteases as novel players in the molecular pathogenesis of SCA17.
Assuntos
Calpaína , Ataxias Espinocerebelares , Animais , Calpaína/genética , Calpaína/metabolismo , Neurônios/metabolismo , Ratos , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de TrinucleotídeosRESUMO
Aberrant O-GlcNAcylation, a protein posttranslational modification defined by the O-linked attachment of the monosaccharide N-acetylglucosamine (O-GlcNAc), has been implicated in neurodegenerative diseases. However, although many neuronal proteins are substrates for O-GlcNAcylation, this process has not been extensively investigated in polyglutamine disorders. We aimed to evaluate the enzyme O-GlcNAc transferase (OGT), which attaches O-GlcNAc to target proteins, in Machado-Joseph disease (MJD). MJD is a neurodegenerative condition characterized by ataxia and caused by the expansion of a polyglutamine stretch within the deubiquitinase ataxin-3, which then present increased propensity to aggregate. By analyzing MJD cell and animal models, we provide evidence that OGT is dysregulated in MJD, therefore compromising the O-GlcNAc cycle. Moreover, we demonstrate that wild-type ataxin-3 modulates OGT protein levels in a proteasome-dependent manner, and we present OGT as a substrate for ataxin-3. Targeting OGT levels and activity reduced ataxin-3 aggregates, improved protein clearance and cell viability, and alleviated motor impairment reminiscent of ataxia of MJD patients in zebrafish model of the disease. Taken together, our results point to a direct interaction between OGT and ataxin-3 in health and disease and propose the O-GlcNAc cycle as a promising target for the development of therapeutics in the yet incurable MJD.
Assuntos
Ataxina-3/metabolismo , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , N-Acetilglucosaminiltransferases/metabolismo , Animais , Ataxina-3/genética , Modelos Animais de Doenças , Células HEK293 , Humanos , Peptídeos , Complexo de Endopeptidases do Proteassoma , Peixe-Zebra/metabolismoRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.
Assuntos
Ataxina-3/fisiologia , Doença de Machado-Joseph/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Peptídeos/metabolismo , Fosfoproteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose , Ataxina-3/genética , Linhagem Celular , Fibroblastos , Quinase 3 da Glicogênio Sintase/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/fisiologia , Análise Serial de Proteínas , Proteínas Proto-Oncogênicas c-akt/fisiologia , Serina-Treonina Quinases TOR/fisiologiaRESUMO
Proteolytic machineries execute vital cellular functions and their disturbances are implicated in diverse medical conditions, including neurodegenerative diseases. Interestingly, calpains, a class of Ca2+-dependent regulatory proteases, can modulate the degradational system of autophagy by cleaving proteins involved in this pathway. Moreover, both machineries are common players in many molecular pathomechanisms and have been targeted individually or together, as a therapeutic strategy in experimental setups. In this review, we briefly introduce calpains and autophagy, with their roles in health and disease, and focus on their direct pathologically relevant interplay in neurodegeneration and beyond. The modulation of calpain activity may comprise a promising treatment approach to attenuate the deregulation of these two essential mechanisms.