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[This corrects the article DOI: 10.1371/journal.pbio.3002355.].
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The introduction of premature termination codons (PTCs), as a result of splicing defects, insertions, deletions, or point mutations (also termed nonsense mutations), lead to numerous genetic diseases, ranging from rare neuro-metabolic disorders to relatively common inheritable cancer syndromes and muscular dystrophies. Over the years, a large number of studies have demonstrated that certain antibiotics and other synthetic molecules can act as PTC suppressors by inducing readthrough of nonsense mutations, thereby restoring the expression of full-length proteins. Unfortunately, most PTC readthrough-inducing agents are toxic, have limited effects, and cannot be used for therapeutic purposes. Thus, further efforts are required to improve the clinical outcome of nonsense mutation suppressors. Here, by focusing on enhancing readthrough of pathogenic nonsense mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, we show that disturbing the protein translation initiation complex, as well as targeting other stages of the protein translation machinery, enhances both antibiotic and non-antibiotic-mediated readthrough of nonsense mutations. These findings strongly increase our understanding of the mechanisms involved in nonsense mutation readthrough and facilitate the development of novel therapeutic targets for nonsense suppression to restore protein expression from a large variety of disease-causing mutated transcripts.
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Códon sem Sentido , Neoplasias , Humanos , Códon sem Sentido/genética , Biossíntese de Proteínas/genética , Antibacterianos/farmacologiaRESUMO
A 4-year-old boy presented with subacute onset of cerebellar ataxia. Neuroimaging revealed cerebellar atrophy. Metabolic screening tests aiming to detect potentially treatable ataxias showed an increased value (fourfold upper limit of normal) for phytanic acid and elevated very-long-chain fatty acid (VLCFA) ratios (C24:0/C22:0 and C26:0/C22:0), while absolute concentrations of VLCFA were normal. Genetic analysis identified biallelic variants in PEX10. Immunohistochemistry confirmed pathogenicity in the patients' cultured fibroblasts demonstrating peroxisomal mosaicism with a general catalase import deficiency as well as conspicuous peroxisome morphology as an expression of impaired peroxisomal function. We describe for the first time an elongated peroxisome morphology in a patient with PEX10-related cerebellar ataxia.A literature search yielded 14 similar patients from nine families with PEX10-related cerebellar ataxia, most of them presenting their first symptoms between 3 and 8 years of age. In 11/14 patients, the first and main symptom was cerebellar ataxia; in three patients, it was sensorineural hearing impairment. Finally, all 14 patients developed ataxia. Polyneuropathy (9/14) and cognitive impairment (9/14) were common associated findings. In 12/13 patients brain MRI showed cerebellar atrophy. Phytanic acid was elevated in 8/12 patients, while absolute concentrations of VLCFA levels were in normal limits in several patients. VLCFA ratios (C24:0/C22:0 and/or C26:0/C22:0), though, were elevated in 11/11 cases. We suggest including measurement of phytanic acid and VLCFA ratios in metabolic screening tests in unexplained autosomal recessive ataxias with cerebellar atrophy, especially when there is an early onset and symptoms are mild.
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Ataxia Cerebelar , Ataxia/genética , Atrofia , Ataxia Cerebelar/diagnóstico , Ataxia Cerebelar/genética , Pré-Escolar , Testes Genéticos , Humanos , Masculino , Peroxinas/genética , Ácido Fitânico , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
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.
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Cálcio , Acoplamento Excitação-Contração , Animais , Disferlina/genética , Camundongos , Miócitos Cardíacos , Ratos , Retículo SarcoplasmáticoRESUMO
Peroxisomes are cellular organelles with vital functions in lipid, amino acid and redox metabolism. The cellular formation and dynamics of peroxisomes are governed by PEX genes; however, the regulation of peroxisome abundance is still poorly understood. Here, we use a high-content microscopy screen in Saccharomyces cerevisiae to identify new regulators of peroxisome size and abundance. Our screen led to the identification of a previously uncharacterized gene, which we term PEX35, which affects peroxisome abundance. PEX35 encodes a peroxisomal membrane protein, a remote homolog to several curvature-generating human proteins. We systematically characterized the genetic and physical interactome as well as the metabolome of mutants in PEX35, and we found that Pex35 functionally interacts with the vesicle-budding-inducer Arf1. Our results highlight the functional interaction between peroxisomes and the secretory pathway.
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Proteínas de Membrana/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Epistasia Genética , Deleção de Genes , Genes Fúngicos , Microscopia , Saccharomyces cerevisiae/genéticaRESUMO
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.
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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/metabolismoRESUMO
In a compartmentalized cell, correct protein localization is crucial for function of virtually all cellular processes. From the cytoplasm as a starting point, proteins are imported into organelles by specific targeting signals. Many proteins, however, act in more than one cellular compartment. In this chapter, we discuss mechanisms by which proteins can be targeted to multiple organelles with a focus on a novel gene regulatory mechanism, functional translational readthrough, that permits multiple targeting of proteins to the peroxisome and other organelles. In mammals, lactate and malate dehydrogenase are the best-characterized enzymes whose targeting is controlled by functional translational readthrough.
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Peroxissomos/metabolismo , Biossíntese de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Animais , Citoplasma/metabolismo , Mamíferos/genética , Mamíferos/metabolismo , Peroxissomos/genética , Transporte Proteico/genéticaRESUMO
Translational readthrough (TR) has come into renewed focus because systems biology approaches have identified the first human genes undergoing functional translational readthrough (FTR). FTR creates functional extensions to proteins by continuing translation of the mRNA downstream of the stop codon. Here we review recent developments in TR research with a focus on the identification of FTR in humans and the systems biology methods that have spurred these discoveries.
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Peroxissomos/genética , Biossíntese de Proteínas/genética , Ribossomos/genética , Biologia de Sistemas , Animais , Códon de Terminação , Drosophila melanogaster/genética , Genômica , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
The peroxisomal targeting signal type 1 (PTS1) is a seemingly simple peptide sequence at the C-terminal end of most peroxisomal matrix proteins. PTS1 can be described as a tripeptide with the consensus motif [S/A/C] [K/R/H] L. However, this description is neither necessary nor sufficient. It does not cover all cases of PTS1 proteins, and some proteins in accordance with this consensus do not target to the peroxisome. In order to find new PTS proteins in yeast and to arrive at a more complete description of the PTS1 consensus motif, we developed a machine learning approach that involves orthologue expansion of the set of known peroxisomal proteins. We performed a genome-wide in silico screen, characterised several PTS1-containing peptides and identified two new peroxisomal matrix proteins, which we named Pxp1 (Yel020c) and Pxp2 (Yjr111c). Based on these in silico and in vivo analyses, we revised the yeast PTS1 consensus which now includes all known PTS1 proteins.
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Aprendizado de Máquina , Peroxissomos/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Motivos de Aminoácidos , Sequência Consenso , Bases de Dados Genéticas , Genoma Fúngico , Estudo de Associação Genômica Ampla , Receptor 1 de Sinal de Orientação para Peroxissomos , Peroxissomos/genética , Sinais Direcionadores de Proteínas/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
In peroxisome formation, models of near-autonomous peroxisome biogenesis with membrane protein integration directly from the cytosol into the peroxisomal membrane are in direct conflict with models whereby peroxisomes bud from the endoplasmic reticulum and receive their membrane proteins through a branch of the secretory pathway. We therefore reinvestigated the role of the Sec61 complex, the protein-conducting channel of the endoplasmic reticulum (ER) in peroxisome formation. We found that depletion or partial inactivation of Sec61 in yeast disables peroxisome formation. The ER entry of the early peroxisomal membrane protein Pex3 engineered with a glycosylation tag is reduced in sec61 mutant cells. Moreover, we were able to reconstitute Pex3 import into ER membranes in vitro, and we identified a variant of a signal anchor sequence for ER translocation at the Pex3 N-terminus. Our findings are consistent with a Sec61 requirement for peroxisome formation and a fundamental role of the ER in peroxisome biogenesis.
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Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Peroxissomos/fisiologia , Saccharomyces cerevisiae/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Sistemas de Liberação de Medicamentos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Peroxinas , Processamento de Proteína Pós-Traducional , Canais de Translocação SEC , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
During budding of yeast cells peroxisomes are distributed over mother cell and bud, a process that involves the myosin motor protein Myo2p and the peroxisomal membrane protein Inp2p. Here, we show that Pex19p, a peroxin implicated in targeting and complex formation of peroxisomal membrane proteins, also plays a role in peroxisome partitioning. Binding studies revealed that Pex19p interacts with the cargo-binding domain of Myo2p. We identified mutations in Myo2p that specifically reduced binding to Pex19p, but not to Inp2p. The interaction between Myo2p and Pex19p was also reduced by a mutation that blocked Pex19p farnesylation. Microscopy revealed that the Pex19p-Myo2p interaction is important for peroxisome inheritance, because mutations that affect this interaction hamper peroxisome inheritance in vivo. Together these data suggest that both Inp2p and Pex19p are required for proper association of peroxisomes to Myo2p.
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Proteínas de Membrana/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Peroxissomos/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Divisão Celular , Proteínas de Membrana/metabolismo , Mutação , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/química , Miosina Tipo V/genética , Peroxissomos/genética , Prenilação , Domínios e Motivos de Interação entre Proteínas , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
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Transtornos Peroxissômicos , Peroxissomos , Humanos , Peroxissomos/genética , Peroxissomos/metabolismo , Ácidos Graxos/metabolismo , Transtornos Peroxissômicos/genética , Transtornos Peroxissômicos/metabolismo , Mitocôndrias/metabolismo , OxirreduçãoRESUMO
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.
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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 HEK293RESUMO
Calcium (Ca2+) is an intracellular messenger that plays an essential role in a variety of cellular processes ranging from early embryonic events to muscle contraction and neuron excitability. Measurement of cytosolic, endoplasmic reticulum (ER), and mitochondrial Ca2+ has contributed immensely to our understanding of cellular physiology. Here we describe the measurement of peroxisomal Ca2+ using ratiometric Ca2+ sensors, enabling measurement of absolute Ca2+ concentration and its dynamics in living cells.
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Cálcio , Retículo Endoplasmático , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Citosol/metabolismoRESUMO
Peroxisomal matrix protein import is facilitated by cycling receptors shuttling between the cytosol and the peroxisomal membrane. One crucial step in this cycle is the ATP-dependent release of the receptors from the peroxisomal membrane. This step is facilitated by the peroxisomal AAA (ATPases associated with various cellular activities) proteins Pex1p and Pex6p with ubiquitination of the receptor being the main signal for its export. Here we report that the AAA complex contains dislocase as well as deubiquitinating activity. Ubp15p, a ubiquitin hydrolase, was identified as a novel constituent of the complex. Ubp15p partially localizes to peroxisomes and is capable of cleaving off ubiquitin moieties from the type I peroxisomal targeting sequence (PTS1) receptor Pex5p. Furthermore, Ubp15p-deficient cells are characterized by a stress-related PTS1 import defect. The results merge into a picture in which removal of ubiquitin from the PTS1 receptor Pex5p is a specific event and might represent a vital step in receptor recycling.
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Endopeptidases/metabolismo , Peroxissomos/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquitina/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Endopeptidases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peroxissomos/genética , Transporte Proteico/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genéticaRESUMO
Here, we report the identification of a novel hydrolase in Saccharomyces cerevisiae. Ldh1p (systematic name, Ybr204cp) comprises the typical GXSXG-type lipase motif of members of the α/ß-hydrolase family and shares some features with the peroxisomal lipase Lpx1p. Both proteins carry a putative peroxisomal targeting signal type1 (PTS1) and can be aligned with two regions of homology. While Lpx1p is known as a peroxisomal enzyme, subcellular localization studies revealed that Ldh1p is predominantly localized to lipid droplets, the storage compartment of nonpolar lipids. Ldh1p is not required for the function and biogenesis of peroxisomes, and targeting of Ldh1p to lipid droplets occurs independently of the PTS1 receptor Pex5p.
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Organelas/metabolismo , Fosfolipases A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Técnicas de Inativação de Genes , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Peroxissomos/metabolismo , Fosfolipases A/química , Fosfolipases A/genética , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Saccharomyces cerevisiae Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.
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Metabolismo dos Lipídeos , Fosfolipases A/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Ensaios Enzimáticos , Esterases/metabolismo , Técnicas de Inativação de Genes , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Tamanho das Organelas/genética , Organelas/metabolismo , Organelas/ultraestrutura , Fosfolipases A/genética , Fosfolipases A/isolamento & purificação , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Alinhamento de SequênciaRESUMO
Calcium is a central signal transduction element in biology. Peroxisomes are essential cellular organelles, yet calcium handling in peroxisomes has been contentious. Recent advances show that peroxisomes are part of calcium homeostasis in cardiac myocytes and therefore may contribute to or even shape their calcium-dependent functionality. However, the mechanisms of calcium movement between peroxisomes and other cellular sites and their mediators remain elusive. Here, we review calcium handling in peroxisomes in concert with other organelles and summarize the most recent knowledge on peroxisomal involvement in calcium dynamics with a focus on mammalian cells.
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DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.
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DNA/química , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Nanotecnologia/métodos , DNA/análise , Corantes Fluorescentes/química , Células HeLa , Humanos , Microscopia ConfocalRESUMO
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.