RESUMO
BACKGROUND: Recently, important discoveries regarding the archaeon that functioned as the "host" in the merger with a bacterium that led to the eukaryotes, its "complex" nature, and its phylogenetic relationship to eukaryotes, have been reported. Based on these new insights proposals have been put forward to get rid of the three-domain Model of life, and replace it with a two-domain model. RESULTS: We present arguments (both regarding timing, complexity, and chemical nature of specific evolutionary processes, as well as regarding genetic structure) to resist such proposals. The three-domain Model represents an accurate description of the differences at the most fundamental level of living organisms, as the eukaryotic lineage that arose from this unique merging event is distinct from both Archaea and Bacteria in a myriad of crucial ways. CONCLUSIONS: We maintain that "a natural system of organisms", as proposed when the three-domain Model of life was introduced, should not be revised when considering the recent discoveries, however exciting they may be.
Assuntos
Archaea/genética , Bactérias/genética , Evolução Biológica , Eucariotos/genética , Classificação , FilogeniaRESUMO
The mammalian skeleton consists of bones that are formed in two different ways: long bones via endochondral ossification and flat bones via intramembranous ossification. These different formation modes may result in differences in the composition of the two bone types. Using the 2D-difference in gel electrophoresis technique and mass spectrometry, we analyzed the composition of murine mineral-associated proteins of calvaria and long bone. Considerable differences in protein composition were observed. Flat bones (calvariae) contained more soluble collagen (8x), pigment epithelium derived factor (3x) and osteoglycin (4x); whereas long bones expressed more chondrocalcin (3x), thrombospondin- 1 (4x), fetuin (4x), secreted phosphoprotein 24 (3x), and thrombin (7x). Although cystatin motifs containing proteins, such as secreted phosphoprotein 24 and fetuin are highly expressed in long bone, they did not inhibit the activity of the cysteine proteinases cathepsin B and K. The solubility of collagen differed which coincided with differences in collagen crosslinking, long bone containing 3x more (hydroxylysine)-pyridinoline. The degradation of long bone collagen by MMP2 (but not by cathepsin K) was impaired. These differences in collagen crosslinking may explain the differences in the proteolytic pathways osteoclasts use to degrade bone. Our data demonstrate considerable differences in protein composition of flat and long bones and strongly suggest functional differences in formation, resorption, and mechanical properties of these bone types.
Assuntos
Fenômenos Biomecânicos/métodos , Reabsorção Óssea , Osso e Ossos/metabolismo , Colágeno/química , Crânio/metabolismo , Animais , Osso e Ossos/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Colágeno Tipo II/metabolismo , Eletroforese em Gel Bidimensional , Proteínas do Olho/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Espectrometria de Massas/métodos , Camundongos , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo , Serpinas/metabolismo , Crânio/fisiologia , Estresse Mecânico , Trombospondinas/metabolismoRESUMO
In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.8 (gBP29) and 26.8 kDa (gBP27) from mitochondria of the insect trypanosome Crithidia fasciculata. gBP29 and gBP27 proved to be similar, Arg + Ala-rich proteins, with pI values of approximately 10.0. gBP27 has no homology to known proteins, but gBP29 is the C.fasciculata orthologue of gBP21 from Trypanosoma brucei, a gRNA-binding protein that associates with active RNA editing complexes. As measured in UV cross-linking assays, His-tagged recombinant gBP29 and gBP27 bind to radiolabelled poly(U) and synthetic gRNAs, while competition experiments suggest a role for the gRNA 3'-(U)-tail in binding to these proteins. Immunoprecipitates of mt extracts generated with antibodies against gBP29 also contained gBP27 and vice versa. The immunoprecipitates further harbored a large proportion of the cellular content of four different gRNAs and of edited and pre-edited NADH dehydrogenase subunit 7 mRNAs, but only small amounts of mt rRNAs. In addition, the bulk of gBP29 and gBP27 co-eluted with gRNAs from gel filtration columns in the high molecular weight range. Together, these results suggest that the proteins are part of a large macromolecular complex(es). We infer that gBP29 and gBP27 are components of the C.fasciculata editing machinery that may interact with gRNAs.
Assuntos
Crithidia fasciculata/genética , Proteínas de Protozoários , Proteínas de Ligação a RNA/genética , RNA/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Dados de Sequência Molecular , Testes de Precipitina , Ligação Proteica , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/genéticaRESUMO
The sequence of subunit 8 of cytochrome c oxidase from Crithidia fasciculata was determined by sequencing cDNA and N-terminus of the mature protein (Mr = 15.7 kDA). The (inferred) protein is homologous to mammalian cox IV and the corresponding cox subunits from yeast, Neurospora crassa and Dictyostelium discoideum, which is reflected in a very similar hydropathy profile. Elements that are conserved in the C. fasciculata sequence include (i) an N-terminal (D/E)-(K/R)-X-K-(X2)-W-(X2)-(I/L) motif, (ii) a putative membrane-spanning region in the middle portion of the protein, and (iii) a C-terminal W-(X13)-(N/D)-P motif. The C. fasciculata protein is synthesized with a cleavable presequence.
Assuntos
Crithidia fasciculata/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Primers do DNA , DNA Complementar , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Substâncias Macromoleculares , Mamíferos , Camundongos , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase. Amino acid sequence analysis, combined with binding studies using dicyclohexyldiimide and azido ATP allowed the identification of two F0 subunits (b and c) and all of the F1 subunits. The F0 b subunit has a low degree of similarity to subunit b from other organisms. The F1 alpha subunit is extremely small making the beta subunit the largest F1 subunit. Other respiratory-chain complexes were also analyzed. Interestingly, an NADH: ubiquinone oxidoreductase (complex I) appeared to be absent, as judged by electron paramagnetic resonance (EPR), enzyme activity and 2D PAGE analysis. Cytochrome c oxidase (complex IV) displayed a subunit pattern identical to that reported for the purified enzyme, whereas cytochrome c reductase (complex III) appeared to contain two extra subunits. A putative complex II was also identified. The amino acid sequences of the subunits of these complexes also show a very low degree of similarity (if any) to the corresponding sequences in other organisms. Remarkably, peptide sequences derived from mitochondrially encoded subunits were not found in spite of the fact that sequences were obtained of virtually all subunits of complex III, IV and V.
Assuntos
Crithidia fasciculata/química , Crithidia fasciculata/genética , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Crithidia fasciculata/enzimologia , Dicicloexilcarbodi-Imida/química , Dicicloexilcarbodi-Imida/metabolismo , Transporte de Elétrons/genética , Eletroforese em Gel Bidimensional , Dados de Sequência Molecular , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Análise de SequênciaRESUMO
Cytochrome c oxidase was purified from the mitochondrial lysate of the insect trypanosomatid Crithidia fasciculata with the aid of a methyl hydrophobic interaction column in a rapid one-step procedure. The purified complex displayed all characteristics expected from a eukaryotic cytochrome c oxidase: the presence of CuA in electron paramagnetic resonance analysis, a characteristic 605 nm peak in reduced-minus-oxidized optical spectroscopy, and the capacity to efficiently oxidize homologous, but not heterologous, cytochrome c. Two-dimensional PAGE showed that C. fasciculata cytochrome c oxidase consists of at least 10 different subunits. N-terminal sequences were obtained from the six smallest subunits of the complex, one of them showing significant similarity to Neurospora crassa cytochrome c oxidase subunit V. The N-terminus of each of the four largest subunits was found to be blocked.
Assuntos
Crithidia fasciculata/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Sequência de Aminoácidos , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Insetos/parasitologia , Cinética , Dados de Sequência Molecular , Conformação Proteica , Análise de Sequência , Homologia de Sequência de Aminoácidos , EspectrofotometriaRESUMO
Oxygen radical formation in mitochondria is an incompletely understood attribute of eukaryotic cells. Recently, a kinetic model was proposed, in which the ratio between electrons entering the respiratory chain via FADH2 or NADH determines radical formation. During glucose breakdown, the ratio is low; during fatty acid breakdown, the ratio is high (the ratio increasing--asymptotically--with fatty acid length to 0.5, when compared with 0.2 for glucose). Thus, fatty acid oxidation would generate higher levels of radical formation. As a result, breakdown of fatty acids, performed without generation of extra FADH2 in mitochondria, could be beneficial for the cell, especially in the case of long and very long chained ones. This possibly has been a major factor in the evolution of peroxisomes. Increased radical formation, as proposed by the model, can also shed light on the lack of neuronal fatty acid oxidation and tells us about hurdles during early eukaryotic evolution. We specifically focus on extending and discussing the model in light of recent publications and findings.
Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Oxirredução , Peroxissomos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transporte de Elétrons/fisiologia , Humanos , Doenças Neurodegenerativas/metabolismoAssuntos
HIV-1 , Latência Viral , Linfócitos T CD4-Positivos , Células Dendríticas , Infecções por HIV , HumanosRESUMO
RNA editing in trypanosomes is the process of insertion and deletion of U residues at specific sites of mitochondrial transcripts mediated by short guide RNAs (gRNAs) that have a 3' oligo(U) extension. Here we describe the identification by UV cross-linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinity for gRNAs, and the characterization of the binding specificity. A 65-kDa protein binds to gRNAs provided they are equipped with a U tail, to post-transcriptionally labelled mitoribosomal 9S and 12S RNAs that also possess a 3' terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23-29 nucleotides. It does not bind to a number of control RNAs, one of which has an internal U stretch of 13 residues. Poly(U), but not poly(C) or total yeast RNA, efficiently competes for binding to gRNA. Proteins of 88 kDa and 30 kDa also bind to gRNAs with a U tail, to mitochondrial ribosomal RNAs and to oligo(U). These proteins, however, require longer oligo(U) for binding (> 39 nucleotides) and they also have an affinity for other U-rich RNAs and poly(C). For comparison, part of the analysis was also carried out with a mitochondrial extract from Trypanosoma brucei. In this organism, gRNA-binding proteins of 83 kDa and 64 kDa were found with the same preference for 3'-terminal oligomeric U stretches as the C. fasciculata 65-kDa protein, whereas the binding specificity of a 26-kDa protein resembled that of the C. fasciculata 88-kDa and 30-kDa proteins. The possible involvement of the proteins in the editing process is discussed.
Assuntos
Proteínas de Transporte/metabolismo , Crithidia fasciculata/metabolismo , Oligorribonucleotídeos/metabolismo , Proteínas de Protozoários/metabolismo , Nucleotídeos de Uracila/metabolismo , Animais , Ligação Competitiva , Proteínas de Transporte/genética , Proteínas de Transporte/efeitos da radiação , Crithidia fasciculata/genética , Reagentes de Ligações Cruzadas , Insetos/parasitologia , Mitocôndrias/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/efeitos da radiação , Edição de RNA , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , Especificidade da Espécie , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Raios UltravioletaRESUMO
We developed an in organello system to label newly synthesized mitochondrially encoded proteins of Trypanosoma brucei. Highly purified mitochondria, prepared under isotonic conditions, were incubated with radioactive methionine and cysteine in a suitable translation buffer. Analysis of mitochondrial extracts on TRIS-Tricine gels revealed a subset of labeled, NP-40-insoluble proteins. The labeling of these proteins was resistant to the cytosol-specific translation inhibitor cycloheximide. The proteins, however, were not labeled in the presence of chloramphenicol or erythromycin, inhibitors of prokaryotic type translation, or puromycin, a general translation inhibitor. These results indicate that isotonically isolated mitochondria of T. brucei are capable of protein synthesis.
Assuntos
Cloranfenicol/farmacologia , Mitocôndrias/metabolismo , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Proteínas de Protozoários/biossíntese , Trypanosoma brucei brucei/metabolismo , Adenosina Trifosfatases/biossíntese , Animais , Mitocôndrias/efeitos dos fármacosRESUMO
Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated. First, purified holo cytochrome c oxidase turned out to be cysteine-rich, in agreement with the high cysteine codon-content of the sequence of mitochondrial cox subunit mRNAs. Second, in mass spectrometry measurements of cytochrome c oxidase, a protein was detected with the predicted molecular weight of cytochrome c oxidase subunit 2. Finally, an antibody generated against a fusion protein produced in Escherichia coli from constructs containing a segment of cytochrome c oxidase subunit 2 cDNA, specifically recognised protein bands present in cytochrome c oxidase following SDS PAGE. However, these proteins were present in the high molecular weight region of the gel, suggesting that cytochrome c oxidase subunit 2 aggregates in the presence of SDS.
Assuntos
Crithidia fasciculata/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Animais , Western Blotting , Crithidia fasciculata/ultraestrutura , Ciclo-Oxigenase 2 , Cisteína/química , Cisteína/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Isoenzimas/química , Isoenzimas/imunologia , Peroxidases/química , Peroxidases/imunologia , Prostaglandina-Endoperóxido Sintases/química , Prostaglandina-Endoperóxido Sintases/imunologia , RNA de Protozoário/biossíntese , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The assembly of cytochrome-c oxidase was studied in human cells cultured in the presence of inhibitors of mitochondrial or cytosolic protein synthesis. Mitochondrial fractions were resolved using two-dimensional PAGE (blue native PAGE and tricine/SDS/PAGE) and subsequent western blots were developed with monoclonal antibodies against specific subunits of cytochrome-c oxidase. Proteins were also visualized using metabolic labeling followed by two-dimensional electrophoresis and fluorography. These techniques allowed identification of two assembly intermediates of cytochrome-c oxidase. Assembly of the 13 subunits of cytochrome-c oxidase starts with the association of subunit I with subunit IV. Then a larger subcomplex is formed, lacking only subunits VIa and either VIIa or VIIb.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/química , Anticorpos Monoclonais , Linhagem Celular , Cicloeximida/farmacologia , Citosol/enzimologia , Doxiciclina/farmacologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Eletroforese em Gel Bidimensional , Estabilidade Enzimática , Humanos , Cinética , Mitocôndrias/enzimologia , Modelos Biológicos , Conformação Proteica , Inibidores da Síntese de Proteínas/farmacologiaRESUMO
With the aid of cDNA and RNA sequence analysis, we have determined to what extent transcripts of mitochondrial maxicircle genes of the insect trypanosome Crithidia fasciculata are altered by RNA editing, a novel mechanism of gene expression which operates via the insertion and deletion of uridine residues. Editing of cytochrome c oxidase (cox) subunit II and III transcripts and of maxicircle unidentified reading frame (MURF) 2 RNA is limited to a small section and results in the creation of a potential AUG translational initiation codon (coxIII, MURF2) or the removal of a frameshift (coxII). No differences with the genomic sequences were observed in the remainder of these RNAs. Surprisingly, NADH dehydrogenase subunit I transcripts were completely unedited in the coding region, implying that an AUG translational initiation codon is absent. The partial ribosomal RNA sequences determined also conform to the gene sequences. Together these results lead to the conclusion that the unusual sequences predicted by the protein and rRNA genes must indeed be present in the gene products. Editing also occurred in the poly(A) tail of RNAs from all protein genes, including those that are unedited in the coding region. The tails display a large variation in AU sequence motifs. Finally, some cDNAs contained sequences absent from both the DNA and the edited RNA. Some of these may represent intermediates in the RNA editing process. We argue, however, that long runs of T may be artefacts of cDNA synthesis.
Assuntos
Crithidia/genética , Regulação da Expressão Gênica , Mitocôndrias/metabolismo , RNA/genética , Transcrição Gênica/genética , Animais , Sequência de Bases , Códon , DNA/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Dados de Sequência Molecular , NADH Desidrogenase/genética , Poli A/genética , Mapeamento por Restrição , UridinaRESUMO
We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem. 227, 780-786).
Assuntos
Mitocôndrias/química , Poli U/metabolismo , Proteínas de Protozoários , Edição de RNA , RNA de Protozoário/biossíntese , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/química , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Dados de Sequência Molecular , Peso Molecular , Ligação Proteica , RNA/metabolismo , RNA Mitocondrial , RNA Ribossômico/biossíntese , Proteínas de Ligação a RNA/genéticaRESUMO
We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8-fold difference between the molar ratio of these two gRNAs relative to the (pre)-mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5' end but with a different 3' uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing-like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3'-terminus to an editing site of pre-edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5' end of pre-edited RNA.
Assuntos
Crithidia fasciculata/genética , Edição de RNA , RNA Guia de Cinetoplastídeos/genética , RNA de Protozoário/genética , Animais , Sequência de Bases , Ligação de Hidrogênio , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genéticaRESUMO
The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.