RESUMO
Leber's hereditary optic neuropathy (LHON) is an inherited mitochondrial disease that usually leads to acute or subacute bilateral central vision loss. In 95% of cases, LHON is caused by one of three primary mutations of the mitochondrial DNA (mtDNA), m.11778G>A in the MT-ND4 gene, m.14484T>C in the MT-ND6 gene, or m.3460G>A in the MT-ND1 gene. Here we characterize clinically, genetically, and biochemically a LHON family with multiple patients harboring two of these primary LHON mutations, m.11778G>A homoplasmic and m.14484T>C heteroplasmic. The unusually low male-to-female ratio of affected family members is also seen among the other patients previously reported with two primary LHON mutations m.11778G>A and m.14484T>C. While the index patient had very late onset of symptoms at 75years and severe visual loss, her two daughters had both onset in childhood (6 and 9years), with moderate to mild visual loss. A higher degree of heteroplasmy of the m.14484T>C mutation was found to correlate with an earlier age at onset in this family. Ours is the first LHON family harboring two primary LHON mutations where functional studies were performed in several affected family members. A more pronounced bioenergetic defect was found to correlate with an earlier age at onset. The patient with the earliest age at onset had a more significant complex I dysfunction than all controls, including the LHON patient with only the m.11778G>A mutation, suggesting a synergistic effect of the two primary LHON mutations in this patient.
Assuntos
DNA Mitocondrial/genética , Saúde da Família , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/patologia , Mutação Puntual , Adulto , Fatores Etários , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Razão de MasculinidadeAssuntos
Delusões/fisiopatologia , Transtornos Paranoides/fisiopatologia , Doença de Tay-Sachs/fisiopatologia , Cadeia alfa da beta-Hexosaminidase/genética , Adulto , Idade de Início , Delusões/etiologia , Feminino , Humanos , Masculino , Mutação , Transtornos Paranoides/etiologia , Irmãos , Doença de Tay-Sachs/complicações , Doença de Tay-Sachs/genética , Adulto JovemRESUMO
Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana.
RESUMO
In most studies, amounts of protein complexes of the oxidative phosphorylation (OXPHOS) system in different organs or tissues are quantified on the basis of isolated mitochondrial fractions. However, yield of mitochondrial isolations might differ with respect to tissue type due to varying efficiencies of cell disruption during organelle isolation procedures or due to tissue-specific properties of organelles. Here we report an immunological investigation on the ratio of the OXPHOS complexes in different tissues of Arabidopsis thaliana which is based on total protein fractions isolated from five Arabidopsis organs (leaves, stems, flowers, roots and seeds) and from callus. Antibodies were generated against one surface exposed subunit of each of the five OXPHOS complexes and used for systematic immunoblotting experiments. Amounts of all complexes are highest in flowers (likewise with respect to organ fresh weight or total protein content of the flower fraction). Relative amounts of protein complexes in all other fractions were determined with respect to their amounts in flowers. Our investigation reveals high relative amounts of complex I in green organs (leaves and stems) but much lower amounts in non-green organs (roots, callus tissue). In contrast, complex II only is represented by low relative amounts in green organs but by significantly higher amounts in non-green organs, especially in seeds. In fact, the complex I-complex II ratio differs by factor 37 between callus and leaf, indicating drastic differences in electron entry into the respiratory chain in these two fractions. Variation in amounts concerning complexes III, IV and V was less pronounced in different Arabidopsis tissues (quantification of complex V in leaves was not meaningful due to a cross-reaction of the antibody with the chloroplast form of this enzyme). Analyses were complemented by in gel activity measurements for the protein complexes of the OXPHOS system and comparative 2D blue native/SDS PAGE analyses using isolated mitochondria. We suggest that complex I has an especially important role in the context of photosynthesis which might be due to its indirect involvement in photorespiration and its numerous enzymatic side activities in plants.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Transporte de Elétrons , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Fosforilação Oxidativa , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Caules de Planta/metabolismoRESUMO
Classically, DIGE is carried out on the basis of two-dimensional (2D) IEF/SDS PAGE. This allows comparative analyses of large protein sets. However, 2D IEF/SDS PAGE only poorly resolves hydrophobic proteins and is not compatible with native protein characterizations. Blue native PAGE represents a powerful alternative. Combined with CyDye labeling, blue native DIGE offers several useful applications like quantitative comparison of protein complexes of related protein fractions. Here we present a protocol for fluorophore labeling of native protein fractions for separation by blue native PAGE.
Assuntos
Proteínas/análise , Corantes de Rosanilina , Eletroforese em Gel Diferencial Bidimensional/métodos , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
The five complexes (complexes I-V) of the oxidative phosphorylation (OXPHOS) system of mitochondria can be extracted in the form of active supercomplexes. Single-particle electron microscopy has provided 2D and 3D data describing the interaction between complexes I and III, among I, III and IV and in a dimeric form of complex V, between two ATP synthase monomers. The stable interactions are called supercomplexes which also form higher-ordered oligomers. Cryo-electron tomography provides new insights on how these supercomplexes are arranged within intact mitochondria. The structure and function of OXPHOS supercomplexes are discussed.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Clorófitas/metabolismo , Tomografia com Microscopia Eletrônica , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Imageamento Tridimensional , Microscopia Eletrônica , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Modelos Moleculares , Fosforilação Oxidativa , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/ultraestrutura , Multimerização ProteicaRESUMO
In contrast to other retroviruses, foamy viruses (FVs) generate their Pol protein precursor independently of the Gag protein from a spliced mRNA. The exact mechanism of Pol protein incorporation into the viral capsid is poorly understood. Previously, we showed that Pol encapsidation critically depends on the packaging of (pre-) genomic RNA and identified two distinct signals within the cis-acting sequences (CASI and CASII), Pol encapsidation sequences (PESI and PESII), which are required for Pol capsid incorporation. Here, we investigated whether the presence of PESI and PESII in an FV vector is sufficient for Pol encapsidation and whether the rather extended CASII element can be shortened without loss of functionality. Our results indicate that (i) the presence of PESI and II are not sufficient for Pol encapsidation, (ii) prototype FV vectors with a shortened CASII element retain Pol incorporation and full functionality, in particular upon transducing fibroblasts and primary human mesenchymal stem cells, (iii) the presence of the central poly purine tract significantly increased the transduction rates of FV vectors and (iv) Pol encapsidation and RNA packaging can be clearly separated. In essence, we designed a new FV vector that bears approximately 850 bp less of CAS than previously established vectors and is fully functional when analysed to transduce cell lines and primary human cells.
Assuntos
Produtos do Gene pol/genética , Vetores Genéticos , Spumavirus/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Primers do DNA , Genoma Viral , Humanos , Rim , Fases de Leitura Aberta , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Viral/genética , Spumavirus/fisiologia , Transfecção , Replicação ViralRESUMO
The projection structures of complex I and the I+III2 supercomplex from the C4 plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 A. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric gamma-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the gamma-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant "type II" particle has a 15 A shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant "type I" particle. The I+III2 supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of "type I". This would mean that the "type II" particles are not involved in the supercomplex formation and, hence, could have a different physiological role.
Assuntos
Anidrases Carbônicas/química , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/química , Zea mays/metabolismo , Sequência de Aminoácidos , Arabidopsis/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Zea mays/químicaRESUMO
Similar to the lentiviruses family of retroviruses, foamy viruses (FVs) contain purine-rich sequences located in the center of the genome. Their function on viral replication or vector transfer remains elusive, although dual initiation of plus-strand reverse transcription has been suggested. To elucidate the physical nature of the central region of the prototype FV (PFV) genome, we performed 3' and 5' RACE experiments. Our results revealed that the PFV genome contains a centrally located gap in the DNA plus-strand with no definite termination and start point and of variable length. We did not find evidence for a DNA flap region. The PFV isolate harbors four centrally located purine-rich elements (A-D). Only the D element is identical in sequence to the 3' poly purine tract (PPT). We mutated these elements while conserving or altering the overlapping pol reading frame and analyzed the mutants for transient replication in an infectious or for vector transfer in a replication-deficient background. In addition, we determined the protein composition of the respective viral particles. The A and B elements appeared to play a role in Pol protein encapsidation, the C element is likely involved in regulating gene expression, while mutation of the D element resulted in an insignificant reduction in transiently replicating virus and an approximately 50% reduction in vector titer. The reason for this deficit remains to be elucidated.
Assuntos
Purinas/química , Spumavirus/genética , Replicação Viral , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , DNA Complementar/genética , DNA Complementar/metabolismo , Regulação Viral da Expressão Gênica , Produtos do Gene pol/metabolismo , Humanos , Dados de Sequência Molecular , Mutação , Poli A/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Transcrição Reversa , Spumavirus/química , Spumavirus/fisiologiaRESUMO
Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76:10069-10073, 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5' long terminal repeat and one at the 3' end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.
Assuntos
Produtos do Gene pol/genética , Produtos do Gene pol/fisiologia , RNA Viral/genética , RNA Viral/metabolismo , Spumavirus/genética , Spumavirus/fisiologia , Linhagem Celular , Mapeamento Cromossômico , Genes Virais , Humanos , Spumavirus/crescimento & desenvolvimento , Proteínas Virais/genética , Proteínas Virais/fisiologia , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/fisiologia , Montagem de Vírus/genética , Montagem de Vírus/fisiologiaRESUMO
Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101.
Assuntos
Produtos do Gene gag/genética , Spumavirus/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Linhagem Celular , Linhagem Celular Tumoral , Sequência Consenso , Produtos do Gene gag/metabolismo , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Spumavirus/patogenicidade , Spumavirus/ultraestrutura , Replicação ViralRESUMO
The foamy virus (FV) Pol polyprotein is translated independently of Gag from a spliced mRNA. This method of expression raises the question of how Pol is associated with the viral particle. Using a transient FV vector transfection system, it is shown that pregenomic RNA is required for efficient virion incorporation of functionally active Pol and that protein-protein interactions of Pol with Gag are not sufficient to complete particle assembly.