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INTRODUCTION: Molecular testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to suffer from delays and shortages. Antigen tests have recently emerged as a viable alternative to detect patients with high viral loads, associated with elevated risk of transmission. While rapid lateral flow tests greatly improved accessibility of SARS-CoV-2 detection in critical areas, their manual nature limits scalability and suitability for large-scale testing schemes. The Elecsys® SARS-CoV-2 Antigen assay allows antigen immunoassays to be carried out on fully automated high-throughput serology platforms. METHODS: A total of 3139 nasopharyngeal and oropharyngeal swabs were collected at 3 different testing sites in Germany. Swab samples were pre-characterized by reverse transcription real-time polymerase chain reaction (RT-qPCR) and consecutively subjected to the antigen immunoassay on either the cobas e 411 or cobas e 801 analyzer. RESULTS: Of the tested respiratory samples, 392 were PCR positive for SARS-CoV-2 RNA. Median concentration was 2.95 × 104 (interquartile range [IQR] 5.1 × 102-3.5 × 106) copies/ml. Overall sensitivity and specificity of the antigen immunoassay were 60.2% (95% confidence interval [CI] 55.2-65.1) and 99.9% (95% CI 99.6-100.0), respectively. A 93.7% (95% CI 89.7-96.5) sensitivity was achieved at a viral RNA concentration ≥ 104 copies/ml (~ cycle threshold [Ct] value < 29.9). CONCLUSION: The Elecsys SARS-CoV-2 Antigen assay reliably detected patient samples with viral loads ≥ 10,000 copies/ml. It thus represents a viable high-throughput alternative for screening of patients or in situations where PCR testing is not readily available.
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BACKGROUND: The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has evoked a pandemic that challenges public health-care systems worldwide. Endothelial cell dysfunction plays a key role in pathophysiology, and simple prognosticators may help to optimize allocation of limited resources. Endothelial activation and stress index (EASIX) is a validated predictor of endothelial complications and outcome after allogeneic stem cell transplantation. Aim of this study was to test if EASIX could predict life-threatening complications in patients with COVID-19. METHODS: SARS-CoV-2-positive, hospitalized patients were enrolled onto a prospective non-interventional register study (n=100). Biomarkers were assessed at hospital admission. Primary endpoint was severe course of disease (mechanical ventilation and/or death, V/D). Results were validated in 126 patients treated in two independent institutions. RESULTS: EASIX at admission was a strong predictor of severe course of the disease (odds ratio for a two-fold change 3.4, 95%CI 1.8-6.3, p<0.001), time to V/D (hazard ratio (HR) for a two-fold change 2.0, 95%CI 1.5-2.6, p<0.001) as well as survival (HR for a two-fold change 1.7, 95%CI 1.2-2.5, p=0.006). The effect was retained in multivariable analysis adjusting for age, gender, and comorbidities and could be validated in the independent cohort. At hospital admission EASIX correlated with increased suppressor of tumorigenicity-2, soluble thrombomodulin, angiopoietin-2, CXCL8, CXCL9 and interleukin-18, but not interferon-alpha. CONCLUSION: EASIX is a validated predictor of COVID19 outcome and an easy-to-access tool to segregate patients in need for intensive surveillance.
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COVID-19/diagnóstico , Células Endoteliales/fisiología , Trasplante de Células Madre Hematopoyéticas , SARS-CoV-2/fisiología , Índice de Severidad de la Enfermedad , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , COVID-19/mortalidad , Femenino , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Respiración Artificial , Análisis de Supervivencia , Trasplante Homólogo , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND AND AIMS: Infectious diseases contribute to stroke risk, and are associated with socioeconomic status (SES). We tested the hypotheses that the aggregate burden of infections increases the risk of ischemic stroke (IS) and partly explains the association between low SES and ischemic stroke. METHODS: In a case-control study with 470 ischemic stroke patients and 809 age- and sex-matched controls, randomly selected from the population, antibodies against the periodontal microbial agents Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, against Chlamydia pneumonia, Mycoplasma pneumoniae (IgA and IgG), and CagA-positive Helicobacter pylori (IgG) were assessed. RESULTS: IgA seropositivity to two microbial agents was significantly associated with IS after adjustment for SES (OR 1.45 95% CI 1.01-2.08), but not in the fully adjusted model (OR 1.32 95% CI 0.86-2.02). By trend, cumulative IgA seropositivity was associated with stroke due to large vessel disease (LVD) after full adjustment (OR 1.88, 95% CI 0.96-3.69). Disadvantageous childhood SES was associated with higher cumulative seropositivity in univariable analyses, however, its strong impact on stroke risk was not influenced by seroepidemiological data in the multivariable model. The strong association between adulthood SES and stroke was rendered nonsignificant when factors of dental care were adjusted for. CONCLUSIONS: Infectious burden assessed with five microbial agents did not independently contribute to ischemic stroke consistently, but may contribute to stroke due to LVD. High infectious burden may not explain the association between childhood SES and stroke risk. Lifestyle factors that include dental negligence may contribute to the association between disadvantageous adulthood SES and stroke.
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Isquemia Encefálica/complicaciones , Infecciones/complicaciones , Clase Social , Accidente Cerebrovascular/complicaciones , Anciano , Anciano de 80 o más Años , Anticuerpos/inmunología , Bacterias/aislamiento & purificación , Isquemia Encefálica/patología , Estudios de Casos y Controles , Femenino , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Estilo de Vida , Masculino , Persona de Mediana Edad , Factores de Riesgo , Accidente Cerebrovascular/patología , Adulto JovenRESUMEN
We report a novel heteroplasmic point mutation G8299A in the gene for mitochondrial tRNA(Lys) in a patient with progressive external ophthalmoplegia complicated by recurrent respiratory insufficiency. Biochemical analysis of respiratory chain complexes in muscle homogenate showed a combined complex I and IV deficiency. The transition does not represent a known neutral polymorphism and affects a position in the tRNA acceptor stem which is conserved in primates, leading to a destabilization of this functionally important domain. In vitro analysis of an essential maturation step of the tRNA transcript indicates the probable pathogenicity of this mutation. We hypothesize that there is a causal relationship between the novel G8299A transition and progressive external ophthalmoplegia with recurrent respiratory failure due to a depressed respiratory drive.
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Oftalmoplejía Externa Progresiva Crónica/genética , Mutación Puntual/genética , ARN de Transferencia de Lisina/genética , ARN/genética , Insuficiencia Respiratoria/genética , Secuencia de Bases , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Oftalmoplejía Externa Progresiva Crónica/complicaciones , Oftalmoplejía Externa Progresiva Crónica/diagnóstico , ARN Mitocondrial , Insuficiencia Respiratoria/complicaciones , Insuficiencia Respiratoria/diagnóstico , Prevención SecundariaRESUMEN
Our knowledge of molecular mechanisms underlying mitochondrial disorders in humans has increased considerably during the past two decades. Mitochondrial encephalomyopathies have sporadically been reported in dogs. However, molecular and biochemical data that would lend credence to the suspected mitochondrial origin are largely missing. This study was aimed to characterise a Leigh-like subacute necrotising encephalopathy (SNE) in Yorkshire Terriers and to shed light on its enzymatic and genetic background. The possible resemblance to SNE in Alaskan Huskies and to human Leigh syndrome (LS) was another focus of interest. Eleven terriers with imaging and/or gross evidence of V-shaped, non-contiguous, cyst-like cavitations in the striatum, thalamus and brain stem were included. Neuropathological examinations focussed on muscle, brain pathology and mitochondrial ultrastructure. Further investigations encompassed respiratory-chain activities and the mitochondrial DNA. In contrast to mild non-specific muscle findings, brain pathology featured the stereotypic triad of necrotising grey matter lesions with relative preservation of neurons in the aforementioned regions, multiple cerebral infarcts, and severe patchy Purkinje-cell degeneration in the cerebellar vermis. Two dogs revealed a reduced activity of respiratory-chain-complexes I and IV. Genetic analyses obtained a neutral tRNA-Leu(UUR) A-G-transition only. Neuropathologically, SNE in Yorkshire Terriers is nearly identical to the Alaskan Husky form and very similar to human LS. This study, for the first time, demonstrated that canine SNE can be associated with a combined respiratory chain defect. Mitochondrial tRNA mutations and large genetic rearrangements were excluded as underlying aetiology. Further studies, amongst relevant candidates, should focus on nuclear encoded transcription and translation factors.
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ADN Mitocondrial/genética , Enfermedades de los Perros , Complejo II de Transporte de Electrones/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Enfermedad de Leigh/veterinaria , Animales , Tronco Encefálico/patología , Cuerpo Estriado/patología , Análisis Mutacional de ADN/métodos , Enfermedades de los Perros/enzimología , Enfermedades de los Perros/genética , Enfermedades de los Perros/patología , Perros , Femenino , Enfermedad de Leigh/enzimología , Enfermedad de Leigh/genética , Enfermedad de Leigh/patología , Masculino , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Mutación , Aminoacil-ARN de Transferencia/genética , Tálamo/patologíaRESUMEN
Many muscular and neurological disorders are associated with mitochondrial dysfunction and are often accompanied by changes in mitochondrial morphology. Mutations in the gene encoding OPA1, a protein required for fusion of mitochondria, are associated with hereditary autosomal dominant optic atrophy type I. Here we show that mitochondrial fragmentation correlates with processing of large isoforms of OPA1 in cybrid cells from a patient with myoclonus epilepsy and ragged-red fibers syndrome and in mouse embryonic fibroblasts harboring an error-prone mitochondrial mtDNA polymerase gamma. Furthermore, processed OPA1 was observed in heart tissue derived from heart-specific TFAM knock-out mice suffering from mitochondrial cardiomyopathy and in skeletal muscles from patients suffering from mitochondrial myopathies such as myopathy encephalopathy lactic acidosis and stroke-like episodes. Dissipation of the mitochondrial membrane potential leads to fast induction of proteolytic processing of OPA1 and concomitant fragmentation of mitochondria. Recovery of mitochondrial fusion depended on protein synthesis and was accompanied by resynthesis of large isoforms of OPA1. Fragmentation of mitochondria was prevented by overexpressing OPA1. Taken together, our data indicate that proteolytic processing of OPA1 has a key role in inducing fragmentation of energetically compromised mitochondria. We present the hypothesis that this pathway regulates mitochondrial morphology and serves as an early response to prevent fusion of dysfunctional mitochondria with the functional mitochondrial network.
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GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Mitocondrias/enzimología , Mitocondrias/patología , Animales , Estudios de Casos y Controles , Línea Celular , ADN Mitocondrial/genética , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Metabolismo Energético , Células HeLa , Proteínas del Grupo de Alta Movilidad/deficiencia , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/genética , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Mutación , Procesamiento Proteico-PostraduccionalRESUMEN
Wolfram syndrome is caused by mutations in WFS1 encoding wolframin, a polytopic membrane protein of the endoplasmic reticulum. Here, we investigated the molecular pathomechanisms of four missense and two truncating mutations in WFS1. Expression in COS-7 cells as well as direct analysis of patient cells revealed that WFS1 mutations lead to drastically reduced steady-state levels of wolframin. All mutations resulted in highly unstable proteins which were delivered to proteasomal degradation. No wolframin aggregates were found in patient cells suggesting that Wolfram syndrome is not a disease of protein aggregation. Rather, WFS1 mutations cause loss-of-function by cellular depletion of wolframin.
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Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mutación Missense/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Termodinámica , Síndrome de Wolfram/genética , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Fibroblastos/citología , Fibroblastos/patología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Estructura Cuaternaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
A first component involved in import into the mitochondrial intermembrane space, named Mia40, has been described recently in yeast. Here, we identified the human MIA40 as a novel and ubiquitously expressed component of human mitochondria. It belongs to a novel protein family whose members share six highly conserved cysteine residues constituting a -CXC-CX9C-CX9C- motif. Human MIA40 is significantly smaller than the fungal protein and lacks the N-terminal extension including a transmembrane region and mitochondrial targeting signal. It forms soluble complexes within the intermembrane space of human mitochondria. Depletion of MIA40 in human cells by RNA interference specifically affected steady-state levels of small and cysteine-containing intermembrane space proteins like DDP1 and TIM10A, suggesting that MIA40 acts along the import pathway into the intermembrane space. Studies on the in vivo redox state of human MIA40 demonstrated that it contains intramolecular disulfide bonds. Thiol-trapping assays revealed the co-existence of different oxidation states of human MIA40 within the cell. Furthermore, we show that the twin -CX9C- motif is specifically required for import and stability of MIA40 in mitochondria. Partial mutation of this motif affects stable accumulation of MIA40 in the intermembrane space, whereas mutation of all cysteine residues in this motif inhibits import in mitochondria. Taken together, we conclude that the biogenesis and function of MIA40 in the mitochondrial intermembrane space is dependent on redox processes involving conserved cysteine residues.
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Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/fisiología , Mutación , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Gel , Cartilla de ADN , Humanos , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Datos de Secuencia Molecular , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Mutations in the X-linked deafness-dystonia peptide 1 (DDP1) gene cause Mohr-Tranebjaerg syndrome (MTS), a rare form of deafness associated with dystonia. In the patient presented here, improvement of dystonic symptoms upon treatment with alcohol and GABAergic substances is demonstrated for the first time.
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Clonazepam/uso terapéutico , Trastornos Distónicos/tratamiento farmacológico , Trastornos Distónicos/genética , Moduladores del GABA/uso terapéutico , Proteínas de Transporte de Membrana/genética , Adulto , Trastornos del Conocimiento/complicaciones , Enfermedades Genéticas Ligadas al Cromosoma X/tratamiento farmacológico , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Pérdida Auditiva Sensorineural/complicaciones , Humanos , Masculino , Trastornos Mentales/complicaciones , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mutación Missense/genética , Mioclonía/complicaciones , Mutación Puntual/genética , SíndromeRESUMEN
We showed that the human respiratory chain is organized in supramolecular assemblies of respiratory chain complexes, the respirasomes. The mitochondrial complexes I (NADH dehydrogenase) and III (cytochrome c reductase) form a stable core respirasome to which complex IV (cytochrome c oxidase) can also bind. An analysis of the state of respirasomes in patients with an isolated deficiency of single complexes provided evidence that the formation of respirasomes is essential for the assembly/stability of complex I, the major entry point of respiratory chain substrates. Genetic alterations leading to a loss of complex III prevented respirasome formation and led to the secondary loss of complex I. Therefore, primary complex III assembly deficiencies presented as combined complex III/I defects. This dependence of complex I assembly/stability on respirasome formation has important implications for the diagnosis of mitochondrial respiratory chain disorders.
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Complejo III de Transporte de Electrones/fisiología , Mitocondrias/fisiología , Citocromos b/genética , Detergentes/farmacología , Transporte de Electrón , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , MutaciónRESUMEN
BACKGROUND: The Rhesus antigen Ew (ISBT designation 004 011) was first described in 1955. It is defined by a specific antibody, but its molecular genetic basis has not yet been resolved. STUDY DESIGN AND METHODS: Two individuals serologically characterized to express the rare Rhesus antigen Ew were analyzed by sequencing of all 10 exons of the RHCE gene. RESULTS: A nucleotide exchange at position 500 (T500A) resulting in a Met167Lys amino acid substitution was found in both individuals. Moreover, we show that an individual carrying the Ew antigen is capable to produce an alloantibody against the wild-type E antigen. CONCLUSION: The single-point mutation T500A in exon 4 of the RHCE gene is a molecular basis of the rare Rhesus antigen Ew.
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Proteínas Sanguíneas/genética , Proteínas de la Membrana/genética , Mutación Puntual , Sistema del Grupo Sanguíneo Rh-Hr/genética , Sustitución de Aminoácidos , Exones , Humanos , Masculino , Polimorfismo GenéticoRESUMEN
Tim9, Tim10a, and Tim10b are members of the family of small Tim proteins located in the intermembrane space of mammalian mitochondria. In yeast, members of this family act along the TIM22 import pathway during import of metabolite carriers and other integral inner membrane proteins. Here, we show that the human small proteins form two distinct hetero-oligomeric complexes. A 70-kDa complex that contains Tim9 and Tim10a and a Tim9-10a-10b that is part of a higher molecular weight assembly of 450 kDa. This distribution among two complexes suggests Tim10b to be the functional homologue of yeast Tim12. Both human complexes are tightly associated with the inner membrane and, compared with yeast, soluble 70-kDa complexes appear to be completely absent in the intermembrane space. Thus, the function of soluble 70-kDa complexes as trans-site receptors for incoming carrier proteins is not conserved from lower to higher eukaryotes. During import, the small Tim complexes directly interact with human adenine nucleotide translocator (ANT) in transit in a metal-dependent manner. For insertion of carrier preproteins into the inner membrane, the human small Tim proteins directly interact with human Tim22, the putative insertion pore of the TIM22 translocase. However, in contrast to yeast, only a small fraction of Tim9-Tim10a-Tim10b complex is in a stable association with Tim22. We conclude that different mechanisms and specific requirements for import and insertion of mammalian carrier preproteins have evolved in higher eukaryotes.
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Proteínas de Transporte de Membrana/metabolismo , Mitocondrias Hepáticas/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Transporte Biológico , Evolución Molecular , Células HeLa , Humanos , Mamíferos , Ratones , Levaduras/metabolismoRESUMEN
Mutations of the WFS1 gene are responsible for Wolfram syndrome, a rare, recessive disorder characterized by early-onset, non-autoimmune diabetes mellitus, optic atrophy and further neurological and endocrinological abnormalities. The WFS1 gene encodes wolframin, a putative multispanning membrane glycoprotein of the endoplasmic reticulum. The function of wolframin is completely unknown. In order to characterize wolframin, we have generated polyclonal antibodies against both hydrophilic termini of the protein. Wolframin was found to be ubiquitously expressed with highest levels in brain, pancreas, heart and insulinoma beta-cell lines. Analysis of the structural features provides experimental evidence that wolframin contains nine transmembrane segments and is embedded in the membrane in an N(cyt)/C(lum) topology. Wolframin assembles into higher molecular weight complexes of approximately 400 kDa in the membrane. Pulse-chase experiments demonstrate that during maturation wolframin is N-glycosylated but lacks proteolytical processing. Moreover, N-glycosylation appears to be essential for the biogenesis and stability of wolframin. Here we investigate, for the first time, the molecular mechanisms that cause loss-of-function of wolframin in affected individuals. In patients harboring nonsense mutations complete absence of the mutated wolframin is caused by instability and rapid decay of WFS1 nonsense transcripts. In a patient carrying a compound heterozygous missense mutation, R629W, we found markedly reduced steady-state levels of wolframin. Pulse-chase experiments of mutant wolframin expressed in COS-7 cells indicated that the R629W mutation leads to instability and strongly reduced half-life of wolframin. Thus, the Wolfram syndrome in patients investigated here is caused by reduced protein dosage rather than dysfunction of the mutant wolframin.
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Proteínas de la Membrana/genética , Síndrome de Wolfram/genética , Animales , Anticuerpos , Células COS , Humanos , Proteínas de la Membrana/química , Mutación , TransfecciónRESUMEN
The Mohr-Tranebjaerg syndrome (MTS) is a rare neurodegenerative disorder characterized by early-onset deafness, dystonia and further neurological abnormalities such as cortical blindness, spasticity, dementia and mental retardation. Causative mutations were identified within the deafness-dystonia peptide (DDP1/TIMM8a) gene on the X-chromosome. The DDP1 protein is located in the intermembrane space of human mitochondria. Here, it acts in a complex together with its partner protein Tim13 in a chaperone-like manner to facilitate the import of nuclear-encoded precursor proteins into the mitochondrial inner membrane. Thus, MTS is a novel type of mitochondrial disorder. To obtain more insight into the pathophysiology of this neurodegenerative disorder, we performed for the first time a comprehensive clinical and functional characterization of a patient suffering from MTS. This patient exhibited a typical combination of deafness, dystonia and visual loss. Sequence analysis of the patient's DDP1 gene revealed a G to C transversion at nucleotide position 38 of the first exon. The mutation affects the ATG start codon, thereby changing methionine to isoleucine (M1I), and leads to a complete absence of the DDP1 protein. In addition, the partner protein Tim13 was found to be significantly reduced, suggesting that Tim13 requires the presence of DDP1 for its stabilization. The assessment of mitochondrial functions showed the enzyme activities of the mitochondrial energy-generating systems to be normal in the muscle biopsy. Structural abnormalities or aggregations of mitochondria were absent. Electron microscopy revealed only a mild neurogenic atrophy. Neurophysiological investigations showed cochlear dysfunction and disturbance of visual pathways. PET and MRI studies revealed a multifocal pattern of neurodegeneration with hypometabolic areas predominantly located over the right striatum and parietal cortex and marked atrophy of the occipital lobes. Although the visual loss is caused predominantly by neurodegeneration of the visual cortex, degeneration of the retina and the optic nerve contributes to the visual impairment. The pathological changes in basal ganglia and sensory cortex demonstrate the disintegration of subcortico-cortical circuits and correlate well with the clinical presentation of multifocal dystonia. The data presented here showed that, in contrast to most of the known mitochondrial disorders, MTS appears not to be associated with a functional defect of the energy generation system of the mitochondria. Whereas the specific mitochondrial dysfunction leading to neuronal loss in MTS remains to be clarified, the electrophysiological and neuroimaging findings allowed the multifocal manifestation of neurodegenerative lesions in MTS to be characterized specifically.
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Sordera/genética , Distonía/genética , Proteínas de Transporte de Membrana , Mutación Missense , Proteínas/genética , Adulto , Sordera/fisiopatología , Progresión de la Enfermedad , Distonía/fisiopatología , Humanos , Masculino , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Síndrome , Trastornos de la Visión/genéticaRESUMEN
Mohr-Tranebjaerg syndrome is a progressive, neurodegenerative disorder caused by loss-of-function mutations in the DDP1/TIMM8A gene. DDP1 belongs to a family of evolutionary conserved proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. They mediate the import and insertion of hydrophobic membrane proteins into the mitochondrial inner membrane. All of them share a conserved Cys(4) metal binding site proposed to be required for the formation of zinc fingers. So far, the only missense mutation known to cause a full-blown clinical phenotype is a C66W exchange directly affecting this Cys(4) motif. Here, we show that the mutant human protein is efficiently imported into mitochondria and sorted into the intermembrane space. In contrast to wild-type DDP1, it does not complement the function of its yeast homologue Tim8. The C66W mutation impairs binding of Zn(2+) ions via the Cys(4) motif. As a consequence, the mutated DDP1 is incorrectly folded and loses its ability to assemble into a hetero-hexameric 70-kDa complex with its cognate partner protein human Tim13. Thus, an assembly defect of DDP1 is the molecular basis of Mohr-Tranebjaerg syndrome in patients carrying the C66W mutation.
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Proteínas Portadoras/fisiología , Proteínas de Transporte de Membrana , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae , Secuencias de Aminoácidos , Animales , Humanos , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mutación , Pliegue de Proteína , Proteínas/química , Proteínas/genética , Conejos , Zinc/metabolismo , Dedos de ZincRESUMEN
Carnitine palmitoyltransferase II (CPT-II) mediates the import of long-chain fatty acids into the mitochondrial matrix for subsequent beta-oxidation. Defects of CPT-II manifest as a severe neonatal hepatocardiomuscular form or as a mild muscular phenotype in early infancy or adolescence. CPT-II deficiency is diagnosed by the determination of enzyme activity in tissues involving the time-dependent conversion of radiolabeled CPT-II substrates (isotope-exchange assays) or the formation of chromogenic reaction products. We have established a mass spectrometric assay (MS/MS) for the determination of CPT-II activity based on the stoichiometric formation of acetylcarnitine in a coupled reaction system. In this single-tube reaction system palmitoylcarnitine is converted by CPT-II to free carnitine, which is subsequently esterified to acetylcarnitine by carnitine acetyltransferase. The formation of acetylcarnitine directly correlates with the CPT-II activity. Comparison of the MS/MS method (y) with our routine spectrophotometric assay (x) revealed a linear regression of y = 0.58x + 0.12 (r = 0.8369). Both assays allow one to unambiguously detect patients with the muscular form of CPT-II deficiency. However, the higher specificity and sensitivity as well as the avoidance of the drawbacks inherent in the use of radiolabeled substrates make this mass spectrometric method most suitable for the determination of CPT-II activity.
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Acetilcarnitina/análisis , Carnitina O-Palmitoiltransferasa/análisis , Espectrometría de Masas/métodos , Palmitoilcarnitina/metabolismo , Acetilcarnitina/metabolismo , Carnitina/metabolismo , Carnitina O-Acetiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/metabolismo , Humanos , Cinética , Músculos/enzimología , Enfermedades Musculares/diagnóstico , Enfermedades Musculares/enzimología , Espectrofotometría/métodosAsunto(s)
Mitocondrias/metabolismo , Animales , Proteínas Portadoras/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Translocasas Mitocondriales de ADP y ATP/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/fisiopatología , Precursores de Proteínas/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , SíndromeRESUMEN
Yeast cells represent a powerful model system in cell biology mainly due to their amenability to genetic manipulations. Increasingly, studies focus on mutant genes resulting in alterations of cellular structures and organelles. To ascertain the phenotypic changes involved, it is often desirable to use the resolving power of electron microscopy. In contrast to higher eukaryotic cells, yeast cells are particularly difficult to preserve mainly due to the presence of a thick cell wall that acts as a barrier against diffusion of fixatives. Although several procedures are targeted to overcome these difficulties, none of them have become established as a standard procedure. As a consequence, electron microscopy is still not used routinely as a tool in yeast cell biology. This prompted us to develop an easy-to-follow protocol for yeast transmission electron microscopy that should be useful in all cases where membrane integrity and organelle morphology is emphasized. One means of making the yeast cytoplasm more attainable to fixation and staining solutions is by enzymatic digestion of the cell wall. Following this approach, we were able to reliably preserve yeast cells and their cellular organelles. Enzymatic treatment with zymolyase 20T to partially remove the yeast cell wall allowed the fixation, preservation, and visualization of the yeast cytoplasm revealing detailed ultrastructure. The advancement of this technique is demonstrated with mitochondria as a model organelle. Our studies on various yeast mutants clearly show the power of the enzymatic digestion technique in visualizing subtle changes of membrane structure and organelle morphology.