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1.
J Mol Med (Berl) ; 102(5): 641-653, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38430393

RESUMO

Rett syndrome (RTT) is a neurodevelopmental disorder resulting from genetic mutations in the methyl CpG binding protein 2 (MeCP2) gene. Specifically, around 35% of RTT patients harbor premature termination codons (PTCs) within the MeCP2 gene due to nonsense mutations. A promising therapeutic avenue for these individuals involves the use of aminoglycosides, which stimulate translational readthrough (TR) by causing stop codons to be interpreted as sense codons. However, the effectiveness of this treatment depends on several factors, including the type of stop codon and the surrounding nucleotides, collectively referred to as the stop codon context (SCC). Here, we develop a high-content reporter system to precisely measure TR efficiency at different SCCs, assess the recovery of the full-length MeCP2 protein, and evaluate its subcellular localization. We have conducted a comprehensive investigation into the intricate relationship between SCC characteristics and TR induction, examining a total of 14 pathogenic MeCP2 nonsense mutations with the aim to advance the prospects of personalized therapy for individuals with RTT. Our results demonstrate that TR induction can successfully restore full-length MeCP2 protein, albeit to varying degrees, contingent upon the SCC and the specific position of the PTC within the MeCP2 mRNA. TR induction can lead to the re-establishment of nuclear localization of MeCP2, indicating the potential restoration of protein functionality. In summary, our findings underscore the significance of SCC-specific approaches in the development of tailored therapies for RTT. By unraveling the relationship between SCC and TR therapy, we pave the way for personalized, individualized treatment strategies that hold promise for improving the lives of individuals affected by this debilitating neurodevelopmental disorder. KEY MESSAGES: The efficiency of readthrough induction at MeCP2 premature termination codons strongly depends on the stop codon context. The position of the premature termination codon on the transcript influences the readthrough inducibility. A new high-content dual reporter assay facilitates the measurement and prediction of readthrough efficiency of specific nucleotide stop contexts. Readthrough induction results in the recovery of full-length MeCP2 and its re-localization to the nucleus. MeCP2 requires only one of its annotated nuclear localization signals.


Assuntos
Códon sem Sentido , Códon de Terminação , Proteína 2 de Ligação a Metil-CpG , Síndrome de Rett , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Humanos , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células HEK293
2.
Biol Direct ; 19(1): 14, 2024 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365851

RESUMO

Peroxisomes are primarily studied in the brain, kidney, and liver due to the conspicuous tissue-specific pathology of peroxisomal biogenesis disorders. In contrast, little is known about the role of peroxisomes in other tissues such as the heart. In this meta-analysis, we explore mitochondrial and peroxisomal gene expression on RNA and protein levels in the brain, heart, kidney, and liver, focusing on lipid metabolism. Further, we evaluate a potential developmental and heart region-dependent specificity of our gene set. We find marginal expression of the enzymes for peroxisomal fatty acid oxidation in cardiac tissue in comparison to the liver or cardiac mitochondrial ß-oxidation. However, the expression of peroxisome biogenesis proteins in the heart is similar to other tissues despite low levels of peroxisomal fatty acid oxidation. Strikingly, peroxisomal targeting signal type 2-containing factors and plasmalogen biosynthesis appear to play a fundamental role in explaining the essential protective and supporting functions of cardiac peroxisomes.


Assuntos
Transtornos Peroxissômicos , Peroxissomos , Humanos , Peroxissomos/genética , Peroxissomos/metabolismo , Ácidos Graxos/metabolismo , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/metabolismo , Mitocôndrias/metabolismo , Oxirredução
3.
Biomolecules ; 11(7)2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34356630

RESUMO

Premature termination codon (PTC) mutations account for approximately 10% of pathogenic variants in monogenic diseases. Stimulation of translational readthrough, also known as stop codon suppression, using translational readthrough-inducing drugs (TRIDs) may serve as a possible therapeutic strategy for the treatment of genetic PTC diseases. One important parameter governing readthrough is the stop codon context (SCC)-the stop codon itself and the nucleotides in the vicinity of the stop codon on the mRNA. However, the quantitative influence of the SCC on treatment outcome and on appropriate drug concentrations are largely unknown. Here, we analyze the readthrough-stimulatory effect of various readthrough-inducing drugs on the SCCs of five common premature termination codon mutations of PEX5 in a sensitive dual reporter system. Mutations in PEX5, encoding the peroxisomal targeting signal 1 receptor, can cause peroxisomal biogenesis disorders of the Zellweger spectrum. We show that the stop context has a strong influence on the levels of readthrough stimulation and impacts the choice of the most effective drug and its concentration. These results highlight potential advantages and the personalized medicine nature of an SCC-based strategy in the therapy of rare diseases.


Assuntos
Códon sem Sentido , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Biossíntese de Proteínas , RNA Mensageiro , Células HeLa , Humanos , Transtornos Peroxissômicos/terapia , Receptor 1 de Sinal de Orientação para Peroxissomos/biossíntese , Receptor 1 de Sinal de Orientação para Peroxissomos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Neurobiol Dis ; 143: 105012, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32653672

RESUMO

Heterozygous mutations in the ATP1A3 gene, coding for an alpha subunit isoform (α3) of Na+/K+-ATPase, are the primary genetic cause for rapid-onset dystonia-parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). Recently, cerebellar ataxia, areflexia, pes cavus, optic atrophy and sensorineural hearing loss (CAPOS), early infantile epileptic encephalopathy (EIEE), childhood rapid onset ataxia (CROA) and relapsing encephalopathy with rapid onset ataxia (RECA) extend the clinical spectrum of ATP1A3 related disorders. AHC and RDP demonstrate distinct clinical features, with AHC symptoms being generally more severe compared to RDP. Currently, it is largely unknown what determines the disease severity, and whether severity is linked to the degree of functional impairment of the α3 subunit. Here we compared the effect of twelve different RDP and AHC specific mutations on the expression and function of the α3 Na+/K+-ATPase in transfected HEK cells and oocytes. All studied mutations led to functional impairment of the pump, as reflected by lower survival rate and reduced pump current. No difference in the extent of impairment, nor in the expression level, was found between the two phenotypes, suggesting that these measures of pump dysfunction do not exclusively determine the disease severity.


Assuntos
Distúrbios Distônicos/genética , Hemiplegia/genética , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Distúrbios Distônicos/metabolismo , Células HEK293 , Hemiplegia/metabolismo , Humanos , Mutação , Xenopus
5.
Europace ; 22(7): 1119-1131, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32572487

RESUMO

AIMS: The multi-C2 domain protein dysferlin localizes to the T-Tubule system of skeletal and heart muscles. In skeletal muscle, dysferlin is known to play a role in membrane repair and in T-tubule biogenesis and maintenance. Dysferlin deficiency manifests as muscular dystrophy of proximal and distal muscles. Cardiomyopathies have been also reported, and some dysferlinopathy mouse models develop cardiac dysfunction under stress. Generally, the role and functional relevance of dysferlin in the heart is not clear. The aim of this study was to analyse the effect of dysferlin deficiency on the transverse-axial tubule system (TATS) structure and on Ca2+ homeostasis in the heart. METHODS AND RESULTS: We studied dysferlin localization in rat and mouse cardiomyocytes by immunofluorescence microscopy. In dysferlin-deficient ventricular mouse cardiomyocytes, we analysed the TATS by live staining and assessed Ca2+ handling by patch-clamp experiments and measurement of Ca2+ transients and Ca2+ sparks. We found increasing co-localization of dysferlin with the L-type Ca2+-channel during TATS development and show that dysferlin deficiency leads to pathological loss of transversal and increase in longitudinal elements (axialization). We detected reduced L-type Ca2+-current (ICa,L) in cardiomyocytes from dysferlin-deficient mice and increased frequency of spontaneous sarcoplasmic reticulum Ca2+ release events resulting in pro-arrhythmic contractions. Moreover, cardiomyocytes from dysferlin-deficient mice showed an impaired response to ß-adrenergic receptor stimulation. CONCLUSIONS: Dysferlin is required for TATS biogenesis and maintenance in the heart by controlling the ratio of transversal and axial membrane elements. Absence of dysferlin leads to defects in Ca2+ homeostasis which may contribute to contractile heart dysfunction in dysferlinopathy patients.


Assuntos
Cálcio , Acoplamento Excitação-Contração , Animais , Disferlina/genética , Camundongos , Miócitos Cardíacos , Ratos , Retículo Sarcoplasmático
6.
Methods Mol Biol ; 1595: 81-92, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28409454

RESUMO

Translational readthrough, the decoding of stop codons as sense codons, leads to C-terminal extension of proteins which may lead to the formation of protein isoforms with distinct properties from the original protein. Two proteins have recently been identified that are targeted to the peroxisome via hidden peroxisomal targeting signals in their readthrough extensions. This noninduced basal translational readthrough can be distinguished from pharmacological induction of readthrough by aminoglycosides or other small molecules, which can be used for the treatment of diseases caused by premature stop (termination) codons (PTCs). Readthrough of both, natural stop codons and PTCs, can be quantified in cell culture using reporter systems. In the present article, we describe two dual reporter systems, based on combined fluorescence/luminescence measurement and flow cytometric fluorescence measurement, respectively. Further, we provide a protocol for a fast and efficient cloning procedure of reporter constructs. The dual reporter systems described here help to analyze the peroxisome-specific isoforms of readthrough enzymes as well as potential readthrough therapeutics.


Assuntos
Códon de Terminação , Expressão Gênica , Genes Reporter , Biossíntese de Proteínas/genética , Células Cultivadas , Citometria de Fluxo/métodos , Medições Luminescentes/métodos
7.
J Cell Sci ; 130(5): 841-852, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28104817

RESUMO

The multi-C2 domain protein dysferlin localizes to the plasma membrane and the T-tubule system in skeletal muscle; however, its physiological mode of action is unknown. Mutations in the DYSF gene lead to autosomal recessive limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Here, we show that dysferlin has membrane tubulating capacity and that it shapes the T-tubule system. Dysferlin tubulates liposomes, generates a T-tubule-like membrane system in non-muscle cells, and links the recruitment of phosphatidylinositol 4,5-bisphosphate to the biogenesis of the T-tubule system. Pathogenic mutant forms interfere with all of these functions, indicating that muscular wasting and dystrophy are caused by the dysferlin mutants' inability to form a functional T-tubule membrane system.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Distrofias Musculares/metabolismo , Sarcolema/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Cálcio/metabolismo , Caveolina 3/metabolismo , Chlorocebus aethiops , Dinaminas/metabolismo , Disferlina , Células HeLa , Humanos , Proteínas de Membrana/deficiência , Camundongos Knockout , Proteínas Musculares/deficiência , Distrofias Musculares/patologia , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Condicionamento Físico Animal , Ligação Proteica , Sarcolema/ultraestrutura , Proteínas Supressoras de Tumor/metabolismo
8.
Open Biol ; 6(11)2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27881739

RESUMO

Translational readthrough gives rise to C-terminally extended proteins, thereby providing the cell with new protein isoforms. These may have different properties from the parental proteins if the extensions contain functional domains. While for most genes amino acid incorporation at the stop codon is far lower than 0.1%, about 4% of malate dehydrogenase (MDH1) is physiologically extended by translational readthrough and the actual ratio of MDH1x (extended protein) to 'normal' MDH1 is dependent on the cell type. In human cells, arginine and tryptophan are co-encoded by the MDH1x UGA stop codon. Readthrough is controlled by the 7-nucleotide high-readthrough stop codon context without contribution of the subsequent 50 nucleotides encoding the extension. All vertebrate MDH1x is directed to peroxisomes via a hidden peroxisomal targeting signal (PTS) in the readthrough extension, which is more highly conserved than the extension of lactate dehydrogenase B. The hidden PTS of non-mammalian MDH1x evolved to be more efficient than the PTS of mammalian MDH1x. These results provide insight into the genetic and functional co-evolution of these dually localized dehydrogenases.


Assuntos
Arginina/metabolismo , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Triptofano/metabolismo , Animais , Linhagem Celular , Códon de Terminação , Código Genético , Células HeLa , Humanos , Peroxissomos/genética , Biossíntese de Proteínas
9.
Elife ; 3: e03640, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25247702

RESUMO

Translational readthrough gives rise to low abundance proteins with C-terminal extensions beyond the stop codon. To identify functional translational readthrough, we estimated the readthrough propensity (RTP) of all stop codon contexts of the human genome by a new regression model in silico, identified a nucleotide consensus motif for high RTP by using this model, and analyzed all readthrough extensions in silico with a new predictor for peroxisomal targeting signal type 1 (PTS1). Lactate dehydrogenase B (LDHB) showed the highest combined RTP and PTS1 probability. Experimentally we show that at least 1.6% of the total cellular LDHB is targeted to the peroxisome by a conserved hidden PTS1. The readthrough-extended lactate dehydrogenase subunit LDHBx can also co-import LDHA, the other LDH subunit, into peroxisomes. Peroxisomal LDH is conserved in mammals and likely contributes to redox equivalent regeneration in peroxisomes.


Assuntos
L-Lactato Desidrogenase/metabolismo , Mamíferos/metabolismo , Peroxissomos/enzimologia , Biossíntese de Proteínas , Animais , Sequência de Bases , Linhagem Celular , Códon de Terminação/genética , Simulação por Computador , Sequência Conservada , Genes Reporter , Genoma , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/genética , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas , Transporte Proteico , Ratos
10.
J Struct Biol ; 175(3): 362-71, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21741480

RESUMO

The yeast peroxisomal hydrolase Lpx1 belongs to the α/ß-hydrolase superfamily. In the absence of Lpx1, yeast peroxisomes show an aberrant vacuolated morphology similar to what is found in peroxisomal disorder patients. Here, we present the crystal structure of Lpx1 determined at a resolution of 1.9 Å. The structure reveals the complete catalytic triad with an unusual location of the acid residue after strand ß6 of the canonical α/ß-hydrolase fold. A four-helix cap domain covers the active site. The interface between the α/ß-hydrolase core and the cap domain forms the potential substrate binding site, which may also comprise the tunnel that leads into the protein interior and widens into a cavity. Two further tunnels connect the active site to the protein surface, potentially facilitating substrate access. Lpx1 is a homodimer. The α/ß-hydrolase core folds of the two protomers form the dimer contact site. Further dimerization contacts arise from the mutual embracement of the cap domain of one protomer by the non-canonical C-terminal helix of the other, resulting in a total buried surface area of some 6000 Ų. The unusual C-terminal helix sticks out from the core fold to which it is connected by an extended flexible loop. We analyzed whether this helix is required for dimerization and for import of the dimer into peroxisomes using biochemical assays in vitro and a microscopy-based interaction assay in mammalian cells. Surprisingly, the C-terminal helix is dispensable for dimerization and dimer import. The unusually robust self-interaction suggests that Lpx1 is imported into peroxisomes as dimer.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Peroxissomos/enzimologia , Fosfolipases A/química , Fosfolipases A/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Cromatografia em Gel , Bases de Dados de Proteínas , Dimerização , Hidrolases/genética , Fosfolipases A/genética , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/genética
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