RESUMO
Pathogenic variants that disrupt human mitochondrial protein synthesis are associated with a clinically heterogeneous group of diseases. Despite an impairment in oxidative phosphorylation being a common phenotype, the underlying molecular pathogenesis is more complex than simply a bioenergetic deficiency. Currently, we have limited mechanistic understanding on the scope by which a primary defect in mitochondrial protein synthesis contributes to organelle dysfunction. Since the proteins encoded in the mitochondrial genome are hydrophobic and need co-translational insertion into a lipid bilayer, responsive quality control mechanisms are required to resolve aberrations that arise with the synthesis of truncated and misfolded proteins. Here, we show that defects in the OXA1L-mediated insertion of MT-ATP6 nascent chains into the mitochondrial inner membrane are rapidly resolved by the AFG3L2 protease complex. Using pathogenic MT-ATP6 variants, we then reveal discrete steps in this quality control mechanism and the differential functional consequences to mitochondrial gene expression. The inherent ability of a given cell type to recognize and resolve impairments in mitochondrial protein synthesis may in part contribute at the molecular level to the wide clinical spectrum of these disorders.
Assuntos
Fosforilação Oxidativa , Biossíntese de Proteínas , Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , FenótipoRESUMO
Dysfunction of mitochondrial translation is an increasingly important molecular cause of human disease, but structural defects of mitochondrial ribosomal subunits are rare. We used next-generation sequencing to identify a homozygous variant in the mitochondrial small ribosomal protein 14 (MRPS14, uS14m) in a patient manifesting with perinatal hypertrophic cardiomyopathy, growth retardation, muscle hypotonia, elevated lactate, dysmorphy and mental retardation. In skeletal muscle and fibroblasts from the patient, there was biochemical deficiency in complex IV of the respiratory chain. In fibroblasts, mitochondrial translation was impaired, and ectopic expression of a wild-type MRPS14 cDNA functionally complemented this defect. Surprisingly, the mutant uS14m was stable and did not affect assembly of the small ribosomal subunit. Instead, structural modeling of the uS14m mutation predicted a disruption to the ribosomal mRNA channel.Collectively, our data demonstrate pathogenic mutations in MRPS14 can manifest as a perinatal-onset mitochondrial hypertrophic cardiomyopathy with a novel molecular pathogenic mechanism that impairs the function of mitochondrial ribosomes during translation elongation or mitochondrial mRNA recruitment rather than assembly.
Assuntos
Cardiomiopatia Hipertrófica/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Ribossômicas/genética , Acidose Láctica/genética , Acidose Láctica/metabolismo , Acidose Láctica/patologia , Sequência de Aminoácidos/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Criança , Pré-Escolar , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Lactente , Recém-Nascido , Mitocôndrias/metabolismo , Doenças Mitocondriais/patologia , Ribossomos Mitocondriais/metabolismo , Ribossomos Mitocondriais/patologia , Mutação , LinhagemRESUMO
In this paper, we focus on the metrological aspects of spectroscopic Mueller ellipsometry-i.e. on the uncertainty estimation of the measurement results. With the help of simulated Mueller matrices, we demonstrate that the commonly used merit functions do not return the correct uncertainty for the measurand under consideration (here shown for the relatively simple case of the geometrical parameter layer thickness for the example system of a SiO2 layer on a Si substrate). We identify the non-optimal treatment of measured and sample- induced depolarization as a reason of this discrepancy. Since depolarization results from sample properties in combination with experimental parameters, it must not be minimized during the parameter fit. Therefore, we propose a new merit function treating this issue differently: It implicitly uses the measured depolarization as a weighting parameter. It is very simple and computationally cheap. It compares for each wavelength the measured Jones matrix elements to Cloude's covariance matrix: â¼∑λ jsim,λHmeas,λ + j sim,λ . Moreover, an extension will be presented which allows us to include the measurement noise into this merit function. With this, reliable statistical uncertainties can be calculated. Except for some pre-processing of the raw data, there is no additional computational cost.
RESUMO
Spinocerebellar ataxia 28 is an autosomal dominant neurodegenerative disorder caused by missense mutations affecting the proteolytic domain of AFG3L2, a major component of the mitochondrial m-AAA protease. However, little is known of the underlying pathogenetic mechanisms or how to treat patients with SCA28. Currently available Afg3l2 mutant mice harbour deletions that lead to severe, early-onset neurological phenotypes that do not faithfully reproduce the late-onset and slowly progressing SCA28 phenotype. Here we describe production and detailed analysis of a new knock-in murine model harbouring an Afg3l2 allele carrying the p.Met665Arg patient-derived mutation. Heterozygous mutant mice developed normally but adult mice showed signs of cerebellar ataxia detectable by beam test. Although cerebellar pathology was negative, electrophysiological analysis showed a trend towards increased spontaneous firing in Purkinje cells from heterozygous mutants with respect to wild-type controls. As homozygous mutants died perinatally with evidence of cardiac atrophy, for each genotype we generated mouse embryonic fibroblasts (MEFs) to investigate mitochondrial function. MEFs from mutant mice showed altered mitochondrial bioenergetics, with decreased basal oxygen consumption rate, ATP synthesis and mitochondrial membrane potential. Mitochondrial network formation and morphology was altered, with greatly reduced expression of fusogenic Opa1 isoforms. Mitochondrial alterations were also detected in cerebella of 18-month-old heterozygous mutants and may be a hallmark of disease. Pharmacological inhibition of de novo mitochondrial protein translation with chloramphenicol caused reversal of mitochondrial morphology in homozygous mutant MEFs, supporting the relevance of mitochondrial proteotoxicity for SCA28 pathogenesis and therapy development.
Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Ataxias Espinocerebelares/congênito , Animais , Feminino , Técnicas de Introdução de Genes , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Proteínas Mitocondriais/metabolismo , Mutação de Sentido Incorreto , Células de Purkinje/fisiologia , Células de Purkinje/ultraestrutura , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologiaRESUMO
Leigh syndrome is a severe infantile encephalopathy with an exceptionally variable genetic background. We studied the exome of a child manifesting with Leigh syndrome at one month of age and progressing to death by the age of 2.4 years, and identified novel compound heterozygous variants in PNPT1, encoding the polynucleotide phosphorylase (PNPase). Expression of the wild type PNPT1 in the subject's myoblasts functionally complemented the defects, and the pathogenicity was further supported by structural predictions and protein and RNA analyses. PNPase is a key enzyme in mitochondrial RNA metabolism, with suggested roles in mitochondrial RNA import and degradation. The variants were predicted to locate in the PNPase active site and disturb the RNA processing activity of the enzyme. The PNPase trimer formation was not affected, but specific RNA processing intermediates derived from mitochondrial transcripts of the ND6 subunit of Complex I, as well as small mRNA fragments, accumulated in the subject's myoblasts. Mitochondrial RNA processing mediated by the degradosome consisting of hSUV3 and PNPase is poorly characterized, and controversy on the role and location of PNPase within human mitochondria exists. Our evidence indicates that PNPase activity is essential for the correct maturation of the ND6 transcripts, and likely for the efficient removal of degradation intermediates. Loss of its activity will result in combined respiratory chain deficiency, and a classic respiratory chain-deficiency-associated disease, Leigh syndrome, indicating an essential role for the enzyme for normal function of the mitochondrial respiratory chain.
Assuntos
Exorribonucleases/genética , Exorribonucleases/metabolismo , Doença de Leigh/genética , Pré-Escolar , Exoma , Exorribonucleases/química , Feminino , Expressão Gênica , Humanos , Lactente , Recém-Nascido , Doença de Leigh/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Polirribonucleotídeo Nucleotidiltransferase , RNA/metabolismo , RNA Mensageiro/metabolismo , RNA MitocondrialRESUMO
Mitochondrial aminoacyl-tRNA synthetases are an important group of disease genes typically underlying either a disorder affecting an isolated tissue or a distinct syndrome. Missense mutations in the mitochondrial seryl-tRNA synthetase gene, SARS2, have been identified in HUPRA syndrome (hyperuricemia, pulmonary hypertension, renal failure in infancy, and alkalosis). We report here a homozygous splicing mutation in SARS2 in a patient with progressive spastic paresis. We show that the mutation leads to diminished levels of the synthetase in patient's fibroblasts. This has a destabilizing effect on the tRNASer(AGY) isoacceptor, but to a lesser degree than in HUPRA syndrome patients. tRNASer(UCN) is largely unaffected in both phenotypes. In conclusion, the level of tRNASer(AGY) instability may be a factor in determining tissue manifestation in patients with SARS2 mutations. This finding exemplifies the sensitivity of the nervous system to partially reduced aminoacylation, which is sufficient in other tissues to maintain respiratory chain function.
Assuntos
Mutação , Paraparesia Espástica/genética , Splicing de RNA , Serina-tRNA Ligase/genética , Criança , Homozigoto , Humanos , Masculino , RNA Mensageiro/metabolismoRESUMO
Organelle biosynthesis is a key requirement for cell growth and division. The regulation of mitochondrial biosynthesis exhibits additional layers of complexity compared with that of other organelles because they contain their own genome and dedicated ribosomes. Maintaining these components requires gene expression to be coordinated between the nucleo-cytoplasmic compartment and mitochondria in order to monitor organelle homeostasis and to integrate the responses to the physiological and developmental demands of the cell. Surprisingly, the parameters that are used to monitor or count mitochondrial abundance are not known, nor are the signalling pathways. Inhibiting the translation on mito-ribosomes genetically or with antibiotics can impair cell proliferation and has been attributed to defects in aerobic energy metabolism, even though proliferating cells rely primarily on glycolysis to fuel their metabolic demands. However, a recent study indicates that mitochondrial translational stress and the rescue mechanisms that relieve this stress cause the defect in cell proliferation and occur before any impairment of oxidative phosphorylation. Therefore, the process of mitochondrial translation in itself appears to be an important checkpoint for the monitoring of mitochondrial homeostasis and might have a role in establishing mitochondrial abundance within a cell. This hypothesis article will explore the evidence supporting a role for mito-ribosomes and translation in a mitochondria-counting mechanism.
Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/biossíntese , Ribossomos/metabolismo , Processos de Crescimento Celular/fisiologia , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Biossíntese de Proteínas , Ribossomos/genética , Transdução de SinaisRESUMO
Mueller matrix spectroscopic ellipsometry becomes increasingly important for determining structural parameters of periodic line gratings. Because of the anisotropic character of gratings, the measured Mueller matrix elements are highly azimuthal angle dependent. Measurement results are interpreted by basic principles of diffraction on gratings. The spectral and azimuthal angle dependent intensity changes are correlated to so-called Rayleigh singularities, i.e., wavelengths where the number of diffraction orders changes. The positions of the Rayleigh singularities are calculated analytically and overlapped with measured spectra of two different types of photomasks with transparent and reflecting substrates. For both types of gratings, the Rayleigh singularities reproduce the contours of the spectra. Increasing grating periods result in a shift of these contours to longer wavelengths. Characteristic differences between the two photomasks are explained by the influence of the transmission orders, which are determined by the substrate transparency.
RESUMO
In most organisms, the mitochondrial genes are transcribed by RNA polymerases related to the single-subunit RNA polymerases of bacteriophages like T3 and T7. In flowering plants, duplication(s) of the RpoTm gene coding for the mitochondrial RNA polymerase (RPOTm) led to the evolution of additional RNA polymerases transcribing genes in plastids (RPOTp) or in both mitochondria and plastids (RPOTmp). Two putative RPOTmp enzymes were previously described to be encoded by the nuclear genes RpoTmp1 and RpoTmp2 in the moss Physcomitrella patens. Here, we report on a third Physcomitrella RpoT gene. We determined the sequence of the cDNA. Comparison of the deduced amino acid sequence with sequences of plant organellar RNA polymerases suggests that this gene encodes a functional phage-type RNA polymerase. The 78 N-terminal amino acids of the putative RNA polymerase were fused to GFP and found to target the fusion protein exclusively to mitochondria in Arabidopsis protoplasts. P. patens is the only known organism to possess three mitochondrial RNA polymerases.
Assuntos
Bryopsida/enzimologia , Bryopsida/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Mitocôndrias/enzimologia , Mitocôndrias/genética , Sequência de Aminoácidos , Transporte Biológico , Bryopsida/classificação , RNA Polimerases Dirigidas por DNA/química , Genes Mitocondriais , Dados de Sequência Molecular , Filogenia , Plastídeos/química , Plastídeos/genética , RNA/química , RNA Mitocondrial , Alinhamento de SequênciaRESUMO
BACKGROUND: The genetic complexity of infantile cardiomyopathies is remarkable, and the importance of mitochondrial translation defects as a causative factor is only starting to be recognised. We investigated the genetic basis for infantile onset recessive hypertrophic cardiomyopathy in two siblings. METHODS AND RESULTS: Analysis of respiratory chain enzymes revealed a combined deficiency of complexes I and IV in the heart and skeletal muscle. Exome sequencing uncovered a homozygous mutation (L156R) in MRPL44 of both siblings. MRPL44 encodes a protein in the large subunit of the mitochondrial ribosome and is suggested to locate in close proximity to the tunnel exit of the yeast mitochondrial ribosome. We found severely reduced MRPL44 levels in the patient's heart, skeletal muscle and fibroblasts suggesting that the missense mutation affected the protein stability. In patient fibroblasts, decreased MRPL44 affected assembly of the large ribosomal subunit and stability of 16S rRNA leading to complex IV deficiency. Despite this assembly defect, de novo mitochondrial translation was only mildly affected in fibroblasts suggesting that MRPL44 may have a function in the assembly/stability of nascent mitochondrial polypeptides exiting the ribosome. Retroviral expression of wild-type MRPL44 in patient fibroblasts rescued the large ribosome assembly defect and COX deficiency. CONCLUSIONS: These findings indicate that mitochondrial ribosomal subunit defects can generate tissue-specific manifestations, such as cardiomyopathy.
Assuntos
Cardiomiopatia Hipertrófica/genética , Exoma , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Mutação , Proteínas Ribossômicas/genética , Adolescente , Sequência de Aminoácidos , Cardiomiopatia Hipertrófica/congênito , Ciclo-Oxigenase 1 , Complexo I de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Exoma/genética , Evolução Fatal , Feminino , Fibroblastos/metabolismo , Humanos , Lactente , Doenças Mitocondriais/congênito , Dados de Sequência Molecular , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Linhagem , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Transcription of mitochondrial genes in animals, fungi, and plants relies on the activity of T3/T7 phage-type RNA polymerases. Two such enzymes, RPOTm and RPOTmp, are present in the mitochondria of eudicotyledonous plants; RPOTmp is additionally found in plastids. We have characterized the transcriptional role of the dual-targeted RNA polymerase in mitochondria of Arabidopsis thaliana. Examination of mitochondrial transcripts in rpoTmp mutants revealed major differences in transcript abundances between wild-type and rpoTmp plants. Decreased levels of specific transcripts were correlated with reduced abundances of the respiratory chain complexes I and IV. Altered transcript levels in rpoTmp were found to result from gene-specific transcriptional changes, establishing that RPOTmp functions in distinct transcriptional processes within mitochondria. Decreased transcription of specific genes in rpoTmp was not associated with changes in promoter utilization; therefore, RPOTmp function is not promoter specific but gene specific. This implies that additional gene-specific elements direct the transcription of a subset of mitochondrial genes by RPOTmp.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Transcrição Gênica , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica de Plantas , Genes Mitocondriais , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Mutagênese Insercional , Regiões Promotoras Genéticas , RNA de Plantas/genéticaRESUMO
A stop codon within the mRNA facilitates coordinated termination of protein synthesis, releasing the nascent polypeptide from the ribosome. This essential step in gene expression is impeded with transcripts lacking a stop codon, generating nonstop ribosome complexes. Here, we use deep sequencing to investigate sources of nonstop mRNAs generated from the human mitochondrial genome. We identify diverse types of nonstop mRNAs on mitochondrial ribosomes that are resistant to translation termination by canonical release factors. Failure to resolve these aberrations by the mitochondrial release factor in rescue (MTRFR) imparts a negative regulatory effect on protein synthesis that is associated with human disease. Our findings reveal a source of underlying noise in mitochondrial gene expression and the importance of responsive ribosome quality control mechanisms for cell fitness and human health.
RESUMO
S-adenosyl-L-methionine-dependent rRNA dimethylases mediate the methylation of two conserved adenosines near the 3' end of the rRNA in the small ribosomal subunits of bacteria, archaea and eukaryotes. Proteins related to this family of dimethylases play an essential role as transcription factors (mtTFBs) in fungal and animal mitochondria. Human mitochondrial rRNA is methylated and human mitochondria contain two related mtTFBs, one proposed to act as rRNA dimethylase, the other as transcription factor. The nuclear genome of Arabidopsis thaliana encodes three dimethylase/mtTFB-like proteins, one of which, Dim1B, is shown here to be imported into mitochondria. Transcription initiation by mitochondrial RNA polymerases appears not to be stimulated by Dim1B in vitro. In line with this finding, phylogenetic analyses revealed Dim1B to be more closely related to a group of eukaryotic non-mitochondrial rRNA dimethylases (Dim1s) than to fungal and animal mtTFBs. We found that Dim1B was capable of substituting the E. coli rRNA dimethylase activity of KsgA. Moreover, we observed methylation of the conserved adenines in the 18S rRNA of Arabidopsis mitochondria; this modification was not detectable in a mutant lacking Dim1B. These data provide evidence: (i) for rRNA methylation in Arabidopsis mitochondria; and (ii) that Dim1B is the enzyme catalyzing this process.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Metiltransferases/metabolismo , Mitocôndrias/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Teste de Complementação Genética , Metiltransferases/genética , Mitocôndrias/genética , Mutagênese Insercional , Mutação , Filogenia , RNA de Plantas/genética , RNA Ribossômico 18S/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
An in situ monitoring setup and process control loop were developed and integrated into a magnetron sputtering coater equipped with a Sentech SE 401 single wavelength ellipsometer, including the engineering of software for in situ process control to enhance production accuracy. By using that software, the system allows direct monitoring of the layer thickness on a moving substrate. It is shown that it is possible to determine the complex index of refraction from the distribution of measurements depending on the layer thickness. A strategy has been developed for in situ reverse thickness engineering of the top layers to compensate measurement errors.
RESUMO
Mitochondrial diseases are clinically and genetically heterogeneous disorders, caused by pathogenic variants in either the nuclear or mitochondrial genome. This heterogeneity is particularly striking for disease caused by variants in mitochondrial DNA-encoded tRNA (mt-tRNA) genes, posing challenges for both the treatment of patients and understanding the molecular pathology. In this review, we consider disease caused by the two most common pathogenic mt-tRNA variants: m.3243A>G (within MT-TL1, encoding mt-tRNALeu(UUR) ) and m.8344A>G (within MT-TK, encoding mt-tRNALys ), which together account for the vast majority of all mt-tRNA-related disease. We compare and contrast the clinical disease they are associated with, as well as their molecular pathologies, and consider what is known about the likely molecular mechanisms of disease. Finally, we discuss the role of mitochondrial-nuclear crosstalk in the manifestation of mt-tRNA-associated disease and how research in this area not only has the potential to uncover molecular mechanisms responsible for the vast clinical heterogeneity associated with these variants but also pave the way to develop treatment options for these devastating diseases.
Assuntos
DNA Mitocondrial , Variação Genética , Mitocôndrias , Doenças Mitocondriais , RNA Mitocondrial , RNA de Transferência de Leucina , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , RNA Mitocondrial/genética , RNA Mitocondrial/metabolismo , RNA de Transferência de Leucina/genética , RNA de Transferência de Leucina/metabolismoRESUMO
Mitochondrial ribosomes (mitoribosomes) are tethered to the mitochondrial inner membrane to facilitate the cotranslational membrane insertion of the synthesized proteins. We report cryo-electron microscopy structures of human mitoribosomes with nascent polypeptide, bound to the insertase oxidase assembly 1-like (OXA1L) through three distinct contact sites. OXA1L binding is correlated with a series of conformational changes in the mitoribosomal large subunit that catalyze the delivery of newly synthesized polypeptides. The mechanism relies on the folding of mL45 inside the exit tunnel, forming two specific constriction sites that would limit helix formation of the nascent chain. A gap is formed between the exit and the membrane, making the newly synthesized proteins accessible. Our data elucidate the basis by which mitoribosomes interact with the OXA1L insertase to couple protein synthesis and membrane delivery.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/biossíntese , Proteínas Mitocondriais/metabolismo , Ribossomos Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Microscopia Crioeletrônica , Complexo IV da Cadeia de Transporte de Elétrons/química , Humanos , Proteínas de Membrana/química , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Ribossomos Mitocondriais/ultraestrutura , Modelos Moleculares , Proteínas Nucleares/química , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Ribossomos/metabolismoRESUMO
BACKGROUND: In mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we searched for the respective genes in the basal angiosperm Nuphar advena. RESULTS: By screening a set of BAC library filters, three RpoT genes were identified. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those known from RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of green fluorescent protein (GFP) exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp gene, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. In contrast to the RpoT of the lycophyte Selaginella and those of the moss Physcomitrella, which are according to phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases. CONCLUSIONS: Nuphar advena encodes two mitochondrial and one plastid phage-type RNAP. Identification of a plastid-localized phage-type RNAP in this basal angiosperm, orthologous to all other RpoTp enzymes of flowering plants, suggests that the duplication event giving rise to a nuclear gene-encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastididal RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar suggesting that its occurrence is an evolutionary novelty of eudicotyledonous plants like Arabidopsis.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Evolução Molecular , Genoma de Planta , Nuphar/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , Genes Mitocondriais , Genes de Plantas , Mitocôndrias/genética , Dados de Sequência Molecular , Filogenia , Plastídeos/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA de Plantas/genéticaRESUMO
Hydroxy urea moieties are introduced as a new class of bradykinin B(1) receptor antagonists. First, the SAR of the lead compound was systematically explored. Subsequent optimization resulted in the identification of several biaryl-based hydroxyurea bradykinin B(1) receptor antagonists with low-nanomolar activity and very high oral bioavailability in the rat.
Assuntos
Antagonistas de Receptor B1 da Bradicinina , Hidroxiureia/química , Hidroxiureia/metabolismo , Receptor B1 da Bradicinina/metabolismo , Animais , Disponibilidade Biológica , Células CACO-2 , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Hidroxiureia/administração & dosagem , Masculino , Ligação Proteica/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Efforts to find new bradykinin B(1) receptor antagonists identified 2-aminobenzimidazole as a novel core. Subsequent transformation into five-membered diaminoheterocycle derivatives and their synthesis and SAR is described. This resulted in compounds with low nanomolar activity.
Assuntos
Benzimidazóis/química , Antagonistas de Receptor B1 da Bradicinina , Benzimidazóis/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular , Diaminas/química , Diaminas/metabolismo , Diaminas/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Compostos Heterocíclicos/química , Compostos Heterocíclicos/metabolismo , Compostos Heterocíclicos/farmacologia , Humanos , Receptor B1 da Bradicinina/metabolismo , Relação Estrutura-AtividadeRESUMO
The synthesis and SAR of two series of bradykinin B(1) receptor antagonists is described. The benzamide moiety proved to be a suitable replacement for the aryl ester functionality of biaryl based antagonists. In addition, it was found that semicarbazides can effectively replace cyclopropyl amino acids. The compounds with the best overall profile were biaryl semicarbazides which display high antagonistic activity, low Caco-2 efflux and high oral bioavailability in the rat.