RESUMO
The mitochondrial DNA of trypanosomatid protozoa consists of a complex, intercatenated network of tens of maxicircles and thousands of minicircles. This structure, called kinetoplast DNA (kDNA), requires numerous proteins and multiprotein complexes for replication, segregation, and transcription. In this study, we used a proteomic approach to identify proteins that are associated with the kDNA network. We identified a novel protein encoded by Tb927.2.6100 that was present in a fraction enriched for kDNA and colocalized the protein with kDNA by fluorescence microscopy. RNA interference (RNAi) knockdown of its expression resulted in a growth defect and changes in the proportion of kinetoplasts and nuclei in the cell population. RNAi also resulted in shrinkage and loss of the kinetoplasts, loss of maxicircle and minicircle components of kDNA at similar rates, and (perhaps secondarily) loss of edited and pre-edited mRNA. These results indicate that the Tb927.2.6100 protein is essential for the maintenance of kDNA.
Assuntos
DNA de Cinetoplasto/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Cromatografia de Afinidade , DNA Mitocondrial/metabolismo , Reação em Cadeia da Polimerase , Transporte Proteico , Interferência de RNA , RNA de Protozoário/metabolismo , Frações Subcelulares/metabolismo , Trypanosoma brucei brucei/citologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk-secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI-TOF/TOF MS and 1D-Gel-LC-MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT-PCR and Western blotting. The 1D-Gel-LC-MS/MS and 2DE-MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC-specific proteins that will help the researchers to understand the molecular events taking place during lactation.
Assuntos
Células Epiteliais/química , Glândulas Mamárias Animais/citologia , Leite/citologia , Proteoma/análise , Animais , Bovinos , Feminino , Lactação/metabolismo , Glândulas Mamárias Animais/metabolismo , Redes e Vias Metabólicas , Mapas de Interação de Proteínas , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Proteoma/químicaRESUMO
The mitochondrial respiratory chain is comprised of four different protein complexes (I-IV), which are responsible for electron transport and generation of proton gradient in the mitochondrial intermembrane space. This proton gradient is then used by F0F1-ATP synthase (complex V) to produce ATP by oxidative phosphorylation. In this study, the respiratory complexes I, II, and III were affinity purified from Trypanosoma brucei procyclic form cells and their composition was determined by mass spectrometry. The results along with those that we previously reported for complexes IV and V showed that the respiratome of Trypanosoma is divergent because many of its proteins are unique to this group of organisms. The studies also identified two mitochondrial subunit proteins of respiratory complex IV that are encoded by edited RNAs. Proteomics data from analyses of complexes purified using numerous tagged component proteins in each of the five complexes were used to generate the first predicted protein-protein interaction network of the Trypanosoma brucei respiratory chain. These results provide the first comprehensive insight into the unique composition of the respiratory complexes in Trypanosoma brucei, an early diverged eukaryotic pathogen.
Assuntos
Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Mapeamento de Interação de Proteínas/métodos , Proteoma/metabolismo , Proteômica/métodos , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei , Animais , Sequência de Bases , Cromatografia de Afinidade , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/química , Complexo II de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/química , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Espectrometria de Massas , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Dados de Sequência Molecular , Mapas de Interação de Proteínas/genética , Proteoma/química , Proteoma/genética , Proteínas de Protozoários/genética , Edição de RNA , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologiaRESUMO
Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite's ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro, as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7.
Assuntos
Subunidades Proteicas/metabolismo , Proteínas de Protozoários/metabolismo , RNA Polimerase I/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Dineínas/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Fatores de Transcrição/isolamento & purificação , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genéticaRESUMO
Mitochondrial (mt) gene expression in Trypanosoma brucei entails multiple types of RNA processing, including polycistronic transcript cleavage, mRNA editing, gRNA oligouridylation, and mRNA polyadenylation, which are catalyzed by various multiprotein complexes. We examined the novel mitochondrial RNA-binding 1 (MRB1) complex that has 16 associated proteins, four of which have motifs suggesting RNA interaction. RNase treatment or the lack of kDNA in mutants resulted in lower MRB1 complex sedimentation in gradients, indicating that MRB1 complex associates with kDNA transcripts. RNAi knockdowns of expression of the Tb10.406.0050 (TbRGGm, RGG motif), Tb927.6.1680 (C2H2 zinc finger), and Tb11.02.5390 (no known motif) MRB1 proteins each inhibited in vitro growth of procyclic form parasites and resulted in cells with abnormal numbers of nuclei. Knockdown of TbRGGm, but not the other two proteins, disrupted the MRB1 complex, indicating that it, but perhaps not the other two, is required for complex assembly and/or stability. The knockdowns resulted in similar but nonidentical patterns of altered in vivo abundances of edited, pre-edited, and preprocessed mt mRNAs, but did not appreciably affect the abundances of mRNAs that do not get edited. These results indicate that MRB1 complex is critical to the processing of mt RNAs, and although its specific function is unknown, it appears essential to parasite viability.
Assuntos
Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Técnicas de Silenciamento de Genes , Proteínas de Protozoários/genética , Interferência de RNA , RNA Mitocondrial , Trypanosoma brucei brucei/genéticaRESUMO
The mitochondrial F(0)F(1) ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F(0)F(1) ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F(1) subunits, three to F(0) subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F(1) alpha subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F(0)F(1)-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/fisiologia , Trypanosoma brucei brucei/enzimologia , Trifosfato de Adenosina/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Técnicas de Silenciamento de Genes , Glucose/metabolismo , Imunoprecipitação , ATPases Mitocondriais Próton-Translocadoras/antagonistas & inibidores , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , Oligomicinas/farmacologia , Subunidades Proteicas/fisiologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/fisiologia , Interferência de RNA , Azida Sódica/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
The uridine insertion/deletion RNA editing of kinetoplastid mitochondrial transcripts is performed by complex machinery involving a number of proteins and multiple protein complexes. Here we describe the effect of silencing of TbRGG1 gene by RNA interference on RNA editing in procyclic stage of Trypanosoma brucei. TbRGG1 is an essential protein for cell growth, the absence of which results in an overall decline of edited mRNAs, while the levels of never-edited RNAs remain unaltered. Repression of TbRGG1 expression has no effect on the 20S editosome and MRP1/2 complex. TAP-tag purification of TbRGG1 coisolated a novel multiprotein complex, and its association was further verified by TAP-tag analyses of two other components of the complex. TbRGG1 interaction with this complex appears to be mediated by RNA. Our results suggest that the TbRGG1 protein functions in stabilizing edited RNAs or editing efficiency and that the associated novel complex may have a role in mitochondrial RNA metabolism. We provisionally name it putative mitochondrial RNA-binding complex 1 (put-MRB complex 1).
Assuntos
Proteínas de Protozoários/metabolismo , RNA de Protozoário/metabolismo , Proteínas de Ligação a RNA/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Sequência de Bases , Primers do DNA/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Proteínas de Protozoários/genética , RNA/genética , RNA/metabolismo , Edição de RNA , Interferência de RNA , Estabilidade de RNA , RNA Mitocondrial , RNA de Protozoário/genética , Proteínas de Ligação a RNA/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
African trypanosomes, early diverged eukaryotes and the agents of sleeping sickness, have several basic cellular processes that are remarkably divergent from those in their mammalian hosts. They have large mitochondria and switch between oxidative phosphorylation and glycolysis as the major pathways for energy generation during their life cycle. We report here the identification and characterization of several multiprotein mitochondrial complexes from procyclic form Trypanosoma brucei. These were identified and purified using a panel of monoclonal antibodies that were generated against a submitochondrial protein fraction and using tandem affinity purification (TAP) tag affinity chromatography and localized within the cells by immunofluorescence. Protein composition analyses by mass spectrometry revealed substantial divergence of oxidoreductase complex from that of other organisms and identified a novel complex that may have a function associated with nucleic acids. The relationship to divergent physiological processes in these pathogens is discussed.
Assuntos
Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Oxirredutases/metabolismo , Trypanosoma brucei brucei/enzimologia , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Antiprotozoários/metabolismo , Western Blotting , Linhagem Celular , Fracionamento Químico , Imunofluorescência , Imunoprecipitação , Proteínas Mitocondriais/química , Complexos Multiproteicos/isolamento & purificação , Oxirredutases/química , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Análise de Sequência de Proteína , Frações Subcelulares/metabolismo , Trypanosoma brucei brucei/citologiaRESUMO
Although eukaryotic mitochondrial (mt) ribosomes evolved from a putative prokaryotic ancestor their compositions vary considerably among organisms. We determined the protein composition of tandem affinity-purified Trypanosoma brucei mt ribosomes by mass spectrometry and identified 133 proteins of which 77 were associated with the large subunit and 56 were associated with the small subunit. Comparisons with bacterial and mammalian mt ribosomal proteins identified T. brucei mt homologs of L2-4, L7/12, L9, L11, L13-17, L20-24, L27-30, L33, L38, L43, L46, L47, L49, L52, S5, S6, S8, S9, S11, S15-18, S29, and S34, although the degree of conservation varied widely. Sequence characteristics of some of the component proteins indicated apparent functions in rRNA modification and processing, protein assembly, and mitochondrial metabolism implying possible additional roles for these proteins. Nevertheless most of the identified proteins have no homology outside Kinetoplastida implying very low conservation and/or a divergent function in kinetoplastid mitochondria.
Assuntos
Mitocôndrias/química , Ribossomos/química , Trypanosoma brucei brucei/química , Algoritmos , Animais , Animais Geneticamente Modificados , Células Cultivadas , Cromatografia de Afinidade , Espectrometria de Massas , Mitocôndrias/metabolismo , Proteínas Ribossômicas/química , Subunidades Ribossômicas/química , Ribossomos/metabolismo , Coloração e Rotulagem/métodos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismoRESUMO
In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans- and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.
Assuntos
Proteoma/metabolismo , Splicing de RNA/fisiologia , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA/metabolismo , Spliceossomos/metabolismo , Trypanosoma brucei brucei/metabolismo , Animais , Biologia Computacional/métodos , Sequência Conservada/fisiologia , Evolução Molecular , Espectrometria de Massas , Estrutura Terciária de Proteína/fisiologia , Proteoma/análise , Proteoma/genética , Proteômica , Proteínas de Protozoários/análise , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteínas/análise , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Especificidade da Espécie , Spliceossomos/genética , Trypanosoma brucei brucei/genética , Proteínas Centrais de snRNP/genética , Proteínas Centrais de snRNP/metabolismoRESUMO
Most mitochondrial mRNAs in kinetoplastids require editing, that is, the posttranscriptional insertion and deletion of uridine nucleotides that are specified by guide RNAs and catalyzed by multiprotein complexes. Recent studies have identified many of the proteins in these complexes, in addition to some of their functions and interactions. Although much remains unknown, a picture of highly organized complexes is emerging that shows that the complex that catalyzes the central steps of editing is partitioned into distinct insertion and deletion editing subcomplexes. These subcomplexes coordinate hundreds of ordered catalytic steps that function to produce a single mature mRNA. The dynamic processes, which might entail interactions among multiprotein complexes and changes in their composition and conformation, remain to be elucidated.
Assuntos
Edição de RNA , Trypanosoma/genética , Animais , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Complexos Multiproteicos/metabolismo , Proteínas de Protozoários/metabolismo , RNA Helicases/metabolismo , RNA Ligase (ATP)/metabolismo , RNA Nucleotidiltransferases/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial , Proteínas de Ligação a RNA/metabolismo , Trypanosoma/enzimologia , Trypanosoma/metabolismoRESUMO
Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane, and matrix; each harboring specific functions and structures. In this study, we used LC-MS/MS to characterize the protein composition of Trypanosoma brucei mitochondrial (mt) membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mt membranes with high confidence, and 106 with moderate-to-low confidence. The sub-cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mt membrane have putative roles in metabolic, energy generating, and transport processes, approximately 50% have no known function. These studies result in a comprehensive profile of the composition and sub-organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mt functions.
Assuntos
Membranas Mitocondriais/química , Proteínas Mitocondriais/análise , Proteoma/análise , Proteínas de Protozoários/análise , Trypanosoma brucei brucei/citologia , Cromatografia Líquida , Proteínas Mitocondriais/isolamento & purificação , Proteoma/isolamento & purificação , Proteínas de Protozoários/isolamento & purificação , Espectrometria de Massas em TandemRESUMO
The composition of the large, single, mitochondrion (mt) of Trypanosoma brucei was characterized by MS (2-D LC-MS/MS and gel-LC-MS/MS) analyses. A total of 2897 proteins representing a substantial proportion of procyclic form cellular proteome were identified, which confirmed the validity of the vast majority of gene predictions. The data also showed that the genes annotated as hypothetical (species specific) were overpredicted and that virtually all genes annotated as hypothetical, unlikely are not expressed. By comparing the MS data with genome sequence, 40 genes were identified that were not previously predicted. The data are placed in a publicly available web-based database (www.TrypsProteome.org). The total mitochondrial proteome is estimated at 1008 proteins, with 401, 196, and 283 assigned to the mt with high, moderate, and lower confidence, respectively. The remaining mitochondrial proteins were estimated by statistical methods although individual assignments could not be made. The identified proteins have predicted roles in macromolecular, metabolic, energy generating, and transport processes providing a comprehensive profile of the protein content and function of the T. brucei mt.
Assuntos
Proteínas Mitocondriais/análise , Proteínas de Protozoários/análise , Trypanosoma brucei brucei/metabolismo , Animais , Fracionamento Celular , Bases de Dados de Proteínas , Internet , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Proteômica , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Reprodutibilidade dos Testes , Análise de Sequência de Proteína , Espectrometria de Massas em Tandem , Trypanosoma brucei brucei/genéticaRESUMO
A mitochondrial inner membrane protein, designated MIX, seems to be essential for cell viability. The deletion of both alleles was not possible, and the deletion of a single allele led to a loss of virulence and aberrant mitochondrial segregation and cell division in Leishmania major. However, the mechanism by which MIX exerts its effect has not been determined. We show here that MIX is also expressed in the mitochondrion of Trypanosoma brucei, and using RNA interference, we found that its loss leads to a phenotype that is similar to that described for Leishmania. The loss of MIX also had a major effect on cytochrome c oxidase activity, on the mitochondrial membrane potential, and on the production of mitochondrial ATP by oxidative phosphorylation. Using a tandem affinity purification tag, we found that MIX is associated with a multiprotein complex that contains subunits of the mitochondrial cytochrome c oxidase complex (respiratory complex IV), the composition of which was characterized in detail. The specific function of MIX is unknown, but it appears to be important for the function of complex IV and for mitochondrial segregation and cell division in T. brucei.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Divisão Celular , Complexo IV da Cadeia de Transporte de Elétrons/genética , Expressão Gênica , Potencial da Membrana Mitocondrial , Proteínas Mitocondriais/genética , Ligação Proteica , Proteínas de Protozoários/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Most mitochondrial (mt) mRNAs in trypanosomes undergo posttranscriptional RNA editing, which inserts and deletes uridines (Us) to produce the mature and functional mRNA. The editing process is catalyzed by multiple enzymatic steps and is carried out by an approximately 20S macromolecular complex, the editosome. Editosomes have been purified from Trypanosoma brucei using various techniques including combinations of column chromatography, gradient sedimentation, monoclonal antibody affinity, and TAP-tag affinity approaches. This article describes in detail the methods for editosome purification and identification of protein components by mass spectrometry analyses. It also describes the methods for isolation and analysis of TAP-tagged mutagenized complexes.
Assuntos
Bioquímica/métodos , Proteínas de Protozoários/análise , Edição de RNA/fisiologia , Trypanosoma brucei brucei/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Cromatografia/métodos , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Mitocôndrias/metabolismo , Complexos Multiproteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/químicaRESUMO
The eukaryotic phylum Apicomplexa encompasses thousands of obligate intracellular parasites of humans and animals with immense socio-economic and health impacts. We sequenced nuclear genomes of Chromera velia and Vitrella brassicaformis, free-living non-parasitic photosynthetic algae closely related to apicomplexans. Proteins from key metabolic pathways and from the endomembrane trafficking systems associated with a free-living lifestyle have been progressively and non-randomly lost during adaptation to parasitism. The free-living ancestor contained a broad repertoire of genes many of which were repurposed for parasitic processes, such as extracellular proteins, components of a motility apparatus, and DNA- and RNA-binding protein families. Based on transcriptome analyses across 36 environmental conditions, Chromera orthologs of apicomplexan invasion-related motility genes were co-regulated with genes encoding the flagellar apparatus, supporting the functional contribution of flagella to the evolution of invasion machinery. This study provides insights into how obligate parasites with diverse life strategies arose from a once free-living phototrophic marine alga.
Assuntos
Alveolados/genética , DNA de Algas/química , DNA de Algas/genética , Evolução Molecular , Análise de Sequência de DNA , Perfilação da Expressão Gênica , Dados de Sequência MolecularRESUMO
RNA editing in kinetoplastids is a type of post-transcriptional processing that changes mitochondrial mRNA sequences by the addition or deletion of uridines. Multiple enzymatic activities, such as endoribonuclease and RNA ligase, are associated with this process and exist in a multienzyme complex. Endonuclease activities from Trypanosoma brucei mitochondrial extracts were fractionated by sequential ion exchange and gel filtration chromatography. The RNA editing specific endonuclease activity co-fractionated with in vitro editing while another endonuclease activity with a different substrate specificity, and the majority of mtRNase P activity fractionated away from the editing activity. The pH, salt, temperature, and Mg(2+) optima of all three endonucleases were determined. All three activities are sensitive to high temperature and protease digestion. In addition, treatment with micrococcal nuclease resulted in partial disruption of the editing complex and decreased pre-cleaved in vitro insertion editing activity, suggesting that both RNA(s) and protein(s) are necessary in the intact functional complex.
Assuntos
Endorribonucleases/metabolismo , Mitocôndrias/enzimologia , Trypanosoma brucei brucei/enzimologia , Animais , Endorribonucleases/isolamento & purificação , Edição de RNA , RNA Catalítico/metabolismo , RNA de Protozoário/metabolismo , Ribonuclease PRESUMO
RNA editing processes kinetoplastid mitochondrial transcripts post-transcriptionally by inserting and deleting uridylates (Us) to produce functional mRNAs. The activities of the RNA ligases in the multienzyme complex (the editosome) that catalyzes editing and of the recombinant proteins were characterized and found to be similar. Ligation of two RNA fragments was enhanced when bridged by a complementary RNA or DNA, which left no gaps or overhangs. An acceptor nucleotide preference of G>U>C>A was observed in the absence of exogenous ATP but U was preferred upon addition of ATP and ligase activity was increased. The substrate specificity and catalytic characteristics indicate that RNA ligase activity contributes to the accuracy of RNA editing.
Assuntos
Polinucleotídeo Ligases/metabolismo , Edição de RNA , Trypanosoma brucei brucei/enzimologia , Animais , Kinetoplastida/enzimologia , Kinetoplastida/genética , Ligases/metabolismo , Mitocôndrias/genética , Nucleotídeos/classificação , Nucleotídeos/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Trypanosoma brucei brucei/genéticaRESUMO
The composition of the editosome, a multi-protein complex that catalyzes uridine insertion and deletion RNA editing to produce mature mitochondrial mRNAs in trypanosomes, was analyzed by mass spectrometry. The editosomes were isolated by column chromatography, glycerol gradient sedimentation, and monoclonal antibody affinity purifications. At least 16 proteins form the catalytic core of the editosome, and additional associated proteins were identified. Analyses of mitochondrial fractions identified several non-editosome proteins and multi-protein complexes. These studies contribute to the functional annotation of T. brucei genome.
Assuntos
Proteínas de Protozoários/análise , Edição de RNA , Trypanosoma brucei brucei/química , Sequência de Aminoácidos , Animais , Western Blotting , Substâncias Macromoleculares , Espectrometria de Massas , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Complexos Multiproteicos , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismoRESUMO
This chapter describes the methods used to purify the RNA-editing complex, to identify the proteins by mass spectrometry, and to demonstrate the functions for some of the proteins.