RESUMO
In Saccharomyces cerevisiae, mitoribosomes are composed of a 54S large subunit (mtLSU) and a 37S small subunit (mtSSU). The two subunits altogether contain 73 mitoribosome proteins (MRPs) and two ribosomal RNAs (rRNAs). Although mitoribosomes preserve some similarities with their bacterial counterparts, they have significantly diverged by acquiring new proteins, protein extensions, and new RNA segments, adapting the mitoribosome to the synthesis of highly hydrophobic membrane proteins. In this study, we investigated the functional relevance of mitochondria-specific protein extensions at the C-terminus (C) or N-terminus (N) present in 19 proteins of the mtLSU. The studied mitochondria-specific extensions consist of long tails and loops extending from globular domains that mainly interact with mitochondria-specific proteins and 21S rRNA moieties extensions. The expression of variants devoid of extensions in uL4 (C), uL5 (N), uL13 (N), uL13 (C), uL16 (C), bL17 (N), bL17 (C), bL21 (24), uL22 (N), uL23 (N), uL23 (C), uL24 (C), bL27 (C), bL28 (N), bL28 (C), uL29 (N), uL29 (C), uL30 (C), bL31 (C), and bL32 (C) did not rescue the mitochondrial protein synthesis capacities and respiratory growth of the respective null mutants. On the contrary, the truncated form of the mitoribosome exit tunnel protein uL24 (N) yields a partially functional mitoribosome. Also, the removal of mitochondria-specific sequences from uL1 (N), uL3 (N), uL16 (N), bL9 (N), bL19 (C), uL29 (C), and bL31 (N) did not affect the mitoribosome function and respiratory growth. The collection of mutants described here provides new means to study and evaluate defective assembly modules in the mitoribosome biogenesis process.
Assuntos
Mitocôndrias , Ribossomos Mitocondriais , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Ribossomos Mitocondriais/química , Ribossomos Mitocondriais/metabolismo , Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Coenzyme Q (CoQ) is an essential molecule that consists of a highly substituted benzene ring attached to a polyprenyl tail anchored in the inner mitochondrial membrane. CoQ transfers electrons from NADH dehydrogenase and succinate dehydrogenase complexes toward ubiquinol-cytochrome c reductase, and that allows aerobic growth of cells. In Saccharomyces cerevisiae, the synthesis of CoQ depends on fourteen proteins Coq1p-Co11p, Yah1p, Arh1p, and Hfd1p. Some of these proteins are components of CoQ synthome. Using ab initio molecular modeling and site-directed mutagenesis, we identified the functional residues of the O-methyltransferase Coq3p, which depends on S-adenosylmethionine for catalysis and is necessary for two O-methylation steps required for CoQ maturation. Conserved residues as well as those that coevolved in the protein structure were found to have important roles in respiratory growth, CoQ biosynthesis, and also in the stability of CoQ synthome proteins. Finally, a multiple sequence alignment showed that S. cerevisiae Coq3p has a 45 amino acid residues insertion that is poorly conserved or absent in oleaginous yeast, cells that can store up to 20% of their dry weight as lipids. These results point to the Coq3p structural determinants of its biological and catalytic function and could contribute to the development of lipid-producing yeast for biotechnology.
Assuntos
Metiltransferases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Membranas Mitocondriais , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The human body experiences physiological changes under microgravity environment that phenocopy aging on Earth. These changes include early onset osteoporosis, skeletal muscle atrophy, cardiac dysfunction, and immunosenescence, and such adaptations to the space environment may pose some risk to crewed missions to Mars. To investigate the effect of microgravity on aging, many model organisms have been used such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mice. Herein we report that the budding yeast Saccharomyces cerevisiae show decreased replicative lifespan (RLS) under simulated microgravity in a clinostat. The reduction of yeast lifespan is not a result of decreased tolerance to heat shock or oxidative stress and could be overcome either by deletion of FOB1 or calorie restriction, two known interventions that extend yeast RLS. Deletion of the sirtuin gene SIR2 worsens the simulated microgravity effect on RLS, and together with the fob1Δ mutant phenotype, it suggests that simulated microgravity augments the formation of extrachromosomal rDNA circles, which accumulate in yeast during aging. We also show that the chronological lifespan in minimal medium was not changed when cells were grown in the clinostat. Our data suggest that the reduction in longevity due to simulated microgravity is conserved in yeast, worms, and flies, and these findings may have potential implications for future crewed missions in space, as well as the use of microgravity as a model for human aging.
Assuntos
Envelhecimento , Saccharomyces cerevisiae/fisiologia , Simulação de Ausência de Peso/efeitos adversos , Restrição Calórica , DNA Ribossômico , Proteínas de Ligação a DNA/genética , Longevidade , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genéticaRESUMO
Human HSP27 is a small heat shock protein that modulates the ability of cells to respond to heat shock and oxidative stress, and also functions as a chaperone independent of ATP, participating in the proteasomal degradation of proteins. The expression of HSP27 is associated with survival in mammalian cells. In cancer cells, it confers resistance to chemotherapy; in neurons, HSP27 has a positive effect on neuronal viability in models of Alzheimer's and Parkinson's diseases. To better understand the mechanism by which HSP27 expression contributes to cell survival, we expressed human HSP27 in the budding yeast Saccharomyces cerevisiae under control of different mutant TEF promoters, that conferred nine levels of graded basal expression, and showed that replicative lifespan and proteasomal activity increase as well as the resistance to oxidative and thermal stresses. The profile of these phenotypes display a dose-response effect characteristic of hormesis, an adaptive phenomenon that is observed when cells are exposed to increasing amounts of stress or toxic substances. The hormetic response correlates with changes in expression levels of HSP27 and also with its oligomeric states when correlated to survival assays. Our results indicate that fine tuning of HSP27 concentration could be used as a strategy for cancer therapy, and also for improving neuronal survival in neurodegenerative diseases.
Assuntos
Proteínas de Choque Térmico HSP27 , Hormese , Saccharomyces cerevisiae , Animais , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Resposta ao Choque Térmico , Humanos , Chaperonas Moleculares , Estresse Oxidativo , Saccharomyces cerevisiae/metabolismoRESUMO
DNA synthesis and homologous recombination can be used to simplify molecular cloning and to make synthetic biology easily accessible (M.J. Czar et al., 2009). However, the design of overlapping DNA fragments to construct large molecules is time-consuming and requires verification of several parameters to ensure that fragment synthesis is attainable, given the restrictions found in chemical synthesis of DNA. OVERFRAG is a web-based tool that generates overlapping DNA fragments to assemble either in yeast cells by Gap Repair (H. Ma et al., 1987) or in vitro by (D.G. Gibson et al., 2009) and In-Fusion (B. Zhu et al., 2007) methods. The fragments generated are suitable for chemical synthesis and molecular assembly. Some possible uses include cDNA cloning, design of chimeric antibodies and synthetic biology applications. Web tool is freely available at http://www.each.usp.br/digiampietri/overfrag.
RESUMO
Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , Proteínas RGS/genética , Proteínas RGS/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Recently, a large body of evidences indicates the existence in the mitochondrial matrix of foci that contain different proteins involved in mitochondrial RNA metabolism. Some of these proteins have a pentatricopeptide repeat motif that constitutes their RNA-binding structures. Here we report that MSC6, a mitochondrial pentatricopeptide protein of unknown function, is a multi copy suppressor of mutations in QRS1/HER2 a component of the trimeric complex that catalyzes the transamidation of glutamyl-tRNAQ to glutaminyl-tRNAQ. This is an essential step in mitochondrial translation because of the lack of a specific mitochondrial aminoacyl glutaminyl-tRNA synthetase. MSC6 over-expression did not abolish translation of an aberrant variant form of Cox2p detected in QRS1/HER2 mutants, arguing against a suppression mechanism that bypasses Qrs1p function. A slight decrement of the mitochondrial translation capacity as well as diminished growth on respiratory carbon sources media for respiratory activity was observed in the msc6 null mutant. Additionally, the msc6 null mutant did not display any impairment in RNA transcription, processing or turnover. We concluded that Msc6p is a mitochondrial matrix protein and further studies are required to indicate the specific function of Msc6p in mitochondrial translation.
Assuntos
Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Alelos , Aminoacil-tRNA Sintetases/química , Regulação Fúngica da Expressão Gênica , Genótipo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Transcrição GênicaRESUMO
Coenzyme Q (Q) is an isoprenylated benzoquinone electron carrier required for electronic transport in the mitochondrial respiratory chain, shuttling electrons from complexes I and II to complex III. Q synthesis requires proteins termed Coq (Coq1-Coq11). Coq7p is part of the multimeric complex involved in Q synthesis catalyzing the hydroxylation of demethoxy-Q6 (DMQ6), the last monooxygenase step in Q synthesis with a catalytic center containing a carboxylate-bridged di-iron at the active site of the enzyme. Here we indicate a group of Coq7p residues that modulate protein activity: D53, R57, V111 and S114. R57, V111 and S114 are very conserved residues; V111 and S114 are present in separated communities of amino acid correlation analysis. The coq7 double mutant V111G/S114A and S114E show respiratory deficiency at non permissive temperature, DMQ6 accumulation and lower content of Q6. Therefore we conclude that phosphomimetic S114E inhibit Coq7p activity, and propose that S114 phosphorylation is required to move a non-structured loop of 25 amino acids between helix 2 and 3, and that affects the di-iron coordination in Coq7p catalytic center.
Assuntos
Membranas Mitocondriais/enzimologia , Modelos Moleculares , Ferroproteínas não Heme/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquinona/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Sequência Conservada , Estabilidade Enzimática , Temperatura Alta/efeitos adversos , Hidroxilação , Membranas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ferroproteínas não Heme/química , Ferroproteínas não Heme/genética , Fosforilação , Filogenia , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de SequênciaRESUMO
Mitochondrial retrograde signaling is a communication pathway between the mitochondrion and the nucleus that regulates the expression of a subset of nuclear genes that codify mitochondrial proteins, mediating cell response to mitochondrial dysfunction. In Saccharomyces cerevisiae, the pathway depends on Rtg1p and Rtg3p, which together form the transcription factor that regulates gene expression, and Rtg2p, an activator of the pathway. Here, we provide novel studies aimed at assessing the functional impact of the lack of RTG-dependent signaling on mitochondrial activity. We show that mutants defective in RTG-dependent retrograde signaling present higher oxygen consumption and reduced hydrogen peroxide release in the stationary phase compared to wild-type cells. Interestingly, RTG mutants are less able to decompose hydrogen peroxide or maintain viability when challenged with hydrogen peroxide. Overall, our results indicate that RTG signaling is involved in the hormetic induction of antioxidant defenses and stress resistance.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adaptação Fisiológica/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Regulação Fúngica da Expressão Gênica , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Estresse Oxidativo , Fosforilação , Transporte Proteico/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Bacterial GatCAB amidotransferases are responsible for the transamidation of mischarged glutamyl-tRNA(Gln) into glutaminyl-tRNA(Gln). Mitochondria matrix also has a multienzymatic complex necessary for the transamidation of glutamyl-tRNA(Gln). Gtf1p, Her2p and Pet112p are the constituents of mitochondrial GatFAB amidotransferase complex. Her2p is subunit A of GatFAB complex, while Gtf1p is subunit F, a connector protein between Pet112p (subunit B) and Her2p. Here we evaluate through molecular modeling and amino acid correlation analysis the HER2 protein family. Localization studies indicated that Her2p is predominantly localized in the mitochondrial outer membrane, but it is also located in the mitochondrial matrix where together with Pet112p and Gtf1p constitutes the GatFAB complex. Finally, HER2 random mutagenesis unveiled important residues that provide thermo stability for the complex and are differently suppressed by overexpression of GTF1 or PET112. For instance, her2/ts11 mutant showed its fermentative growth impaired, and poorly rescued by GTF1 indicating that Her2p unknown function in the mitochondria outer membrane affects cell viability.
Assuntos
Aminoacil-tRNA Sintetases/genética , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Transferases de Grupos Nitrogenados/genética , Transferases de Grupos Nitrogenados/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminases/genética , Aminoacil-tRNA Sintetases/metabolismo , Sobrevivência Celular , Mapeamento Cromossômico , Retículo Endoplasmático/metabolismo , Glutamina/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Mutação , Aminoacil-RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Transaminases/metabolismoRESUMO
Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90°C for 1h at pH from 8 to 11.5, while maintaining its biological activity.
Assuntos
Proteínas Recombinantes/química , Superóxido Dismutase/química , Trichoderma/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Clonagem Molecular , Escherichia coli/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Conformação Proteica , Estrutura Secundária de Proteína , Espécies Reativas de Oxigênio , TemperaturaRESUMO
COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q(2). Rescue of respiration by Q(2) is a characteristic of mutants blocked in coenzyme Q(6) synthesis. Unlike Q(6) deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q(6). The physiological significance of earlier observations that purified Coq10p contains bound Q(6) was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q(2). This suggests that in vivo binding of Q(6) by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.
Assuntos
Respiração Celular , Mitocôndrias/enzimologia , Saccharomyces cerevisiae/enzimologia , Ubiquinona/análogos & derivados , Transporte de Elétrons , Deleção de Genes , Saccharomyces cerevisiae/genética , Ubiquinona/biossíntese , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
Deletion of COQ10 in Saccharomyces cerevisiae elicits a respiratory defect characterized by the absence of cytochrome c reduction, which is correctable by the addition of exogenous diffusible coenzyme Q(2). Unlike other coq mutants with hampered coenzyme Q(6) (Q(6) ) synthesis, coq10 mutants have near wild-type concentrations of Q(6). In the present study, we used Q-cycle inhibitors of the coenzyme QH(2)-cytochrome c reductase complex to assess the electron transfer properties of coq10 cells. Our results show that coq10 mutants respond to antimycin A, indicating an active Q-cycle in these mutants, even though they are unable to transport electrons through cytochrome c and are not responsive to myxothiazol. EPR spectroscopic analysis also suggests that wild-type and coq10 mitochondria accumulate similar amounts of Q(6) semiquinone, despite a lower steady-state level of coenzyme QH(2)-cytochrome c reductase complex in the coq10 cells. Confirming the reduced respiratory chain state in coq10 cells, we found that the expression of the Aspergillus fumigatus alternative oxidase in these cells leads to a decrease in antimycin-dependent H(2)O(2) release and improves their respiratory growth.
Assuntos
Antimicina A/farmacologia , Mutação , Saccharomyces cerevisiae/genética , Ubiquinona/análogos & derivados , Antifúngicos/farmacologia , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Grupo dos Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Immunoblotting , Metacrilatos/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxirredução/efeitos dos fármacos , Oxirredutases/genética , Oxirredutases/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Proteínas de Plantas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Superóxidos/metabolismo , Tiazóis/farmacologia , Ubiquinona/genética , Ubiquinona/metabolismoRESUMO
The IL-10 family of cytokines is comprised of IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and IFN-lambdas (IL-28A, IL-28B, and IL-29). The IL-10 family members bind to shared class II cytokine receptor chains that associate in various combinations in heterodimeric complexes. Upon interleukin/receptor complex formation, these proteins switch on the Jak/STAT pathway and elicit pleiotropic biological responses whose variety sharply contrasts with their structural similarities. IL-10 family members are involved in several human diseases and health conditions and hence their structural analyses may provide valuable information to design specific therapeutic strategies. In this review, we describe the human interleukin-10 family of cytokines, focusing on their structures and functions, with particular attention given to IL-22 and IL-10. We report on the recently published structures of IL-10 cytokine family members and their complexes with cognate transmembrane and soluble receptors as well as on interleukin physiology and physiopathology.
Assuntos
Interleucina-10/química , Interleucina-10/metabolismo , Interleucinas/química , Interleucinas/metabolismo , Modelos Moleculares , Receptores de Citocinas/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Humanos , Interleucina-10/classificação , Dados de Sequência Molecular , Filogenia , Multimerização Proteica , Alinhamento de Sequência , Interleucina 22RESUMO
Coq10p is a protein required for coenzyme Q function, but its specific role is still unknown. It is a member of the START domain superfamily that contains a hydrophobic tunnel implicated in the binding of lipophilic molecules. We used site-directed mutagenesis, statistical coupling analysis and molecular modeling to probe structural determinants in the Coq10p putative tunnel. Four point mutations were generated (coq10-K50E, coq10-L96S, coq10-E105K and coq10-K162D) and their biochemical properties analysed, as well as structural consequences. Our results show that all mutations impaired Coq10p function and together with molecular modeling indicate an important role for the Coq10p putative tunnel.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ubiquinona/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Respiração Celular , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Mutação , Conformação ProteicaRESUMO
Interleukin-22 (IL-22) is a cytokine that regulates the production of acute phase proteins of the immunological response. On binding to its cognate receptor (IL-22R1), which is associated to the interleukin-10 receptor 2 (IL-10R2), IL-22 promotes activation of signal transducer and activator of transcription (STAT) pathway and several other cellular responses. A soluble receptor termed interleukin-22 binding protein (IL-22BP) is also able to bind to IL-22 as a natural protein antagonist, and probably provides systemic regulation of IL-22 activity. This inflammatory response system is analyzed here in terms of its molecular physiology and structural assembly. Three-dimensional (3D) model of IL-22 and structural basis of its interactions with the cognate receptors are discussed.
Assuntos
Interleucinas/química , Interleucinas/imunologia , Animais , Bovinos , Cristalografia por Raios X , Cães , Elefantes , Humanos , Inflamação/imunologia , Macaca mulatta , Camundongos , Dados de Sequência Molecular , Pan troglodytes , Ligação Proteica , Conformação Proteica , Ratos , Receptores de Interleucina/imunologia , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Suínos , Interleucina 22RESUMO
Genes for the enzymes that metabolize galactose in Saccharomyces cerevisiae are strongly induced by galactose and tightly repressed by glucose. Because glucose also represses mitochondrial activity, we examined if derepression of the GAL1 galactokinase gene requires physiologically active mitochondria. The effect of mitochondria on the expression of GAL1 was analyzed by a novel approach in which the activity of the organelles was altered by functional expression of URF13, a mitochondrial protein unique to the Texas-type cytoplasmic male sterility phenotype in maize. Mitochondrial targeting and functional expression of the URF13 protein in yeast result in a decrease of the mitochondrial membrane potential similar to those observed in cells treated with mitochondrial inhibitors such as antimycin A or sodium azide. Activation of URF13 in galactose-induced cells results in the inhibition of GAL1 expression in the absence of repressing concentrations of glucose. Our data reveal the existence of a regulatory pathway that connects the derepression of the GAL1 gene with mitochondrial activity.
Assuntos
Galactoquinase/biossíntese , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/metabolismo , Antimicina A/farmacologia , Regulação para Baixo , Galactoquinase/genética , Galactose/metabolismo , Glucose/metabolismo , Potenciais da Membrana , Membranas Mitocondriais/fisiologia , Proteínas Mitocondriais/genética , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Zea maysRESUMO
Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.
Assuntos
Etiquetas de Sequências Expressas , Glucose/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Trichoderma/metabolismo , Acetatos/metabolismo , Núcleo Celular/metabolismo , Ciclo do Ácido Cítrico , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Etanol/metabolismo , Biblioteca Gênica , Glucose/farmacologia , Modelos Biológicos , Dados de Sequência Molecular , NAD/metabolismo , Oxigênio/metabolismo , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Fatores de TempoRESUMO
O gene nuclear GAL1 de Saccharomyces cerevisiae codifica uma galactoquinase induzida por galactose e reprimida por glicose. Três evidências indicam que a transcrição de GAL1 é dependente da atividade mitocondrial. Linhagens petite, com deleção no DNA da organela (pî) ou rompimento em gene nuclear, que codifica a farnesil transferase mitocondrial, são incapazes de induzir GAL1. Os inibidores de respiração antimicina-A e azoteto de sódio (NaN3), que atuam, respectivamente, nos complexos III e IV da cadeia de transporte de elétrons, impedem a indução de GAL1. Em células crescidas em glicose ou glicerol, o oligômero formado pela proteína URF13, na presença de metomil, produz um poro na membrana mitocondrial...