RESUMO
In Arabidopsis, multisubunit RNA polymerases IV and V orchestrate RNA-directed DNA methylation (RdDM) and transcriptional silencing, but what identifies the loci to be silenced is unclear. We show that heritable silent locus identity at a specific subset of RdDM targets requires HISTONE DEACETYLASE 6 (HDA6) acting upstream of Pol IV recruitment and siRNA biogenesis. At these loci, epigenetic memory conferring silent locus identity is erased in hda6 mutants such that restoration of HDA6 activity cannot restore siRNA biogenesis or silencing. Silent locus identity is similarly lost in mutants for the cytosine maintenance methyltransferase, MET1. By contrast, pol IV or pol V mutants disrupt silencing without erasing silent locus identity, allowing restoration of Pol IV or Pol V function to restore silencing. Collectively, these observations indicate that silent locus specification and silencing are separable steps that together account for epigenetic inheritance of the silenced state.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , Epigênese Genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Histona Desacetilases/genética , Interferência de RNA , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Elementos de DNA Transponíveis , RNA Polimerases Dirigidas por DNA/metabolismo , Loci Gênicos , Genótipo , Hereditariedade , Histona Desacetilases/metabolismo , Mutação , Fenótipo , RNA Interferente Pequeno/biossínteseRESUMO
Multisubunit RNA polymerases IV and V (Pols IV and V) mediate RNA-directed DNA methylation and transcriptional silencing of retrotransposons and heterochromatic repeats in plants. We identified genomic sites of Pol V occupancy in parallel with siRNA deep sequencing and methylcytosine mapping, comparing wild-type plants with mutants defective for Pol IV, Pol V, or both Pols IV and V. Approximately 60% of Pol V-associated regions encompass regions of 24-nucleotide (nt) siRNA complementarity and cytosine methylation, consistent with cytosine methylation being guided by base-pairing of Pol IV-dependent siRNAs with Pol V transcripts. However, 27% of Pol V peaks do not overlap sites of 24-nt siRNA biogenesis or cytosine methylation, indicating that Pol V alone does not specify sites of cytosine methylation. Surprisingly, the number of methylated CHH motifs, a hallmark of RNA-directed de novo methylation, is similar in wild-type plants and Pol IV or Pol V mutants. In the mutants, methylation is lost at 50%-60% of the CHH sites that are methylated in the wild type but is gained at new CHH positions, primarily in pericentromeric regions. These results indicate that Pol IV and Pol V are not required for cytosine methyltransferase activity but shape the epigenome by guiding CHH methylation to specific genomic sites.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Citosina/metabolismo , Metilação de DNA , RNA Polimerases Dirigidas por DNA , Genoma de Planta , RNA Interferente Pequeno/metabolismo , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , RNA Interferente Pequeno/genéticaRESUMO
Nuclear multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved in plants as specialized forms of Pol II. Their functions are best understood in the context of RNA-directed DNA methylation (RdDM), a process in which Pol IV-dependent 24 nt siRNAs direct the de novo cytosine methylation of regions transcribed by Pol V. Pol V has additional functions, independent of Pol IV and 24 nt siRNA biogenesis, in maintaining the repression of transposons and genomic repeats whose silencing depends on maintenance cytosine methylation. Here we report that Pol IV and Pol V play unexpected roles in defining the 3' boundaries of Pol II transcription units. Nuclear run-on assays reveal that in the absence of Pol IV or Pol V, Pol II occupancy downstream of poly A sites increases for approximately 12% of protein-coding genes. This effect is most pronounced for convergently transcribed gene pairs. Although Pols IV and V are detected near transcript ends of the affected Pol II - transcribed genes, their role in limiting Pol II read-through is independent of siRNA biogenesis or cytosine methylation for the majority of these genes. Interestingly, we observed that splicing was less efficient in pol IV or pol V mutant plants, compared to wild-type plants, suggesting that Pol IV or Pol V might affect pre-mRNA processing. We speculate that Pols IV and V (and/or their associated factors) play roles in Pol II transcription termination and pre-mRNA splicing by influencing polymerase elongation rates and/or release at collision sites for convergent genes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Arabidopsis/genética , Imunoprecipitação da Cromatina , Metilação de DNA , DNA de Plantas/química , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Mutação , RNA Polimerase II/metabolismo , Splicing de RNA , RNA de Plantas/metabolismo , Análise de Sequência de RNA/métodosRESUMO
The adaptor protein Amot130 scaffolds components of the Hippo pathway to promote the inhibition of cell growth. This study describes how Amot130 through binding and activating the ubiquitin ligase AIP4/Itch achieves these effects. AIP4 is found to bind and ubiquitinate Amot130 at residue Lys-481. This both stabilizes Amot130 and promotes its residence at the plasma membrane. Furthermore, Amot130 is shown to scaffold a complex containing overexpressed AIP4 and the transcriptional co-activator Yes-associated protein (YAP). Consequently, Amot130 promotes the ubiquitination of YAP by AIP4 and prevents AIP4 from binding to large tumor suppressor 1. Amot130 is found to reduce YAP stability. Importantly, Amot130 inhibition of YAP dependent transcription is reversed by AIP4 silencing, whereas Amot130 and AIP4 expression interdependently suppress cell growth. Thus, Amot130 repurposes AIP4 from its previously described role in degrading large tumor suppressor 1 to the inhibition of YAP and cell growth.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Angiomotinas , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Proteínas dos Microfilamentos , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Repressoras/genética , Fatores de Transcrição , Transcrição Gênica/fisiologia , Ubiquitina-Proteína Ligases/genética , Proteínas de Sinalização YAPRESUMO
Gentamicin is a highly efficacious antibiotic against Gram-negative bacteria. However, its usefulness in treating infections is compromised by its poorly understood renal toxicity. Toxic effects are also seen in a variety of other organisms. While the yeast Saccharomyces cerevisiae is relatively insensitive to gentamicin, mutations in any one of â¼20 genes cause a dramatic decrease in resistance. Many of these genes encode proteins important for translation termination or specific protein-trafficking complexes. Subsequent inspection of the physical and genetic interactions of the remaining gentamicin-sensitive mutants revealed a network centered on chitin synthase and the Arf GTPases. Further analysis has demonstrated that some conditional arf1 and gea1 alleles make cells hypersensitive to gentamicin under permissive conditions. These results suggest that one consequence of gentamicin exposure is disruption of Arf-dependent protein trafficking.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Gentamicinas/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/metabolismo , Fator 1 de Ribosilação do ADP/genética , Fator 1 de Ribosilação do ADP/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transporte Proteico/genética , Ratos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Ubiquitination plays an important role in many cellular processes and is implicated in many diseases. Experimental identification of ubiquitination sites is challenging due to rapid turnover of ubiquitinated proteins and the large size of the ubiquitin modifier. We identified 141 new ubiquitination sites using a combination of liquid chromatography, mass spectrometry, and mutant yeast strains. Investigation of the sequence biases and structural preferences around known ubiquitination sites indicated that their properties were similar to those of intrinsically disordered protein regions. Using a combined set of new and previously known ubiquitination sites, we developed a random forest predictor of ubiquitination sites, UbPred. The class-balanced accuracy of UbPred reached 72%, with the area under the ROC curve at 80%. The application of UbPred showed that high confidence Rsp5 ubiquitin ligase substrates and proteins with very short half-lives were significantly enriched in the number of predicted ubiquitination sites. Proteome-wide prediction of ubiquitination sites in Saccharomyces cerevisiae indicated that highly ubiquitinated substrates were prevalent among transcription/enzyme regulators and proteins involved in cell cycle control. In the human proteome, cytoskeletal, cell cycle, regulatory, and cancer-associated proteins display higher extent of ubiquitination than proteins from other functional categories. We show that gain and loss of predicted ubiquitination sites may likely represent a molecular mechanism behind a number of disease-associatedmutations. UbPred is available at http://www.ubpred.org.
Assuntos
Proteoma/análise , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/metabolismo , Proteínas Ubiquitinadas/análise , Sequência de Aminoácidos , Bases de Dados de Proteínas , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de Proteína , Complexos Ubiquitina-Proteína Ligase/metabolismo , Proteínas Ubiquitinadas/metabolismo , UbiquitinaçãoRESUMO
One of the several still unexplained aspects of the mechanism by which the Cdc34/SCF RING-type ubiquitin ligases work is the marked stimulation of Cdc34 autoubiquitination, a phenomenon of unknown mechanism and significance. In in vitro experiments with single-lysine-containing Cdc34 mutant proteins of Saccharomyces cerevisiae, we found that the SCF-mediated stimulation of autoubiquitination is limited to specific N-terminal lysines modified via an intermolecular mechanism. In a striking contrast, SCF quenches autoubiquitination of C-terminal lysines catalyzed in an intramolecular manner. Unlike autoubiquitination of the C-terminal lysines, which has no functional consequence, autoubiquitination of the N-terminal lysines inhibits Cdc34. This autoinhibitory mechanism plays a nonessential role in the catalytic cycle, as the lysineless (K0)Cdc34(DeltaC) is indistinguishable from Cdc34(DeltaC) in ubiquitination of the prototype SCF(Cdc4) substrate Sic1 in vitro, and replacement of the CDC34 gene with either the (K0)cdc34(DeltaC) or the cdc34(DeltaC) allele in yeast has no cell cycle phenotype. We discuss the implications of these findings for the mechanism of Cdc34 function with SCF.
Assuntos
Regulação para Baixo/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Saccharomyces cerevisiae/enzimologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Sítios de Ligação , Catálise , Lisina/metabolismo , Regiões Promotoras Genéticas/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae , Enzimas de Conjugação de Ubiquitina , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/isolamento & purificaçãoRESUMO
[This corrects the article DOI: 10.1186/1747-1028-6-7.].
RESUMO
Decrease in cardiac contractility is a hallmark of chronic diabetes. Previously we showed that this defect results, at least in part, from a dysfunction of the type 2 ryanodine receptor calcium-release channel (RyR2). The mechanism(s) underlying RyR2 dysfunction is not fully understood. The present study was designed to determine whether non-cross-linking advanced glycation end products (AGEs) on RyR2 increase with chronic diabetes and if formation of these post-translational complexes could be attenuated with insulin treatment. Overnight digestion of RyR2 from 8-week control animals (8C) with trypsin afforded 298 peptides with monoisotopic mass (M+H(+)) >or=500. Digestion of RyR2 from 8-week streptozotocin-induced diabetic animals (8D) afforded 21% fewer peptides, whereas RyR2 from 6-week diabetic/2-week insulin-treated animals generated 304 peptides. Using an in-house PERLscript algorithm, search of matrix-assisted laser desorption ionization-time of flight mass data files identified several M+H(+) peaks corresponding to theoretical RyR2 peptides with single N(epsilon)-(carboxymethyl)-lysine, imidazolone A, imidazone B, pyrraline, or 1-alkyl-2-formyl-3,4-glycosyl pyrrole modification that were present in 8D but not 8C. Insulin treatment minimized production of some of these nonenzymatic glycation products. These data show for the first time that AGEs are formed on intracellular RyR2 during diabetes. Because AGE complexes are known to compromise protein activity, these data suggest a potential mechanism for diabetes-induced RyR2 dysfunction.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Produtos Finais de Glicação Avançada/metabolismo , Coração/fisiopatologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica/fisiologia , Masculino , Miocárdio/metabolismo , Fragmentos de Peptídeos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , TripsinaRESUMO
Advanced glycation end products (AGEs), which are composed of various glucose or carbohydrate adducts, are thought to be responsible for several diabetic and age-related complications. However, to date, specific sites on proteins that are modified by AGEs remain largely unknown. We report here the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to determine the type and localization of several AGEs formed in vitro on human beta-2-microglobulin (beta2M), and in vivo on type 2 ryanodine receptor calcium-release channel (RyR2), and sarco(endo)plasmic reticulum (SERCA2a). A PERL script algorithm, developed in-house, makes searching the relatively large amount of data generated by the MALDI-MS more manageable. The outstanding sensitivity of MALDI-TOF-MS coupled with the PERL script algorithm allows such an approach to be a very useful tool in detecting AGEs and other post-translational modifications. We believe that this method could be an important tool when searching for post-translational modifications on proteins.
Assuntos
Algoritmos , Produtos Finais de Glicação Avançada/química , Proteínas/química , Animais , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/isolamento & purificação , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/isolamento & purificação , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Microglobulina beta-2/química , Microglobulina beta-2/genética , Microglobulina beta-2/isolamento & purificaçãoRESUMO
Cell division is controlled in part by the timely activation of the CDK, Cdc28, through its association with G1 and G2 cyclins. Cdc28 complexes are regulated in turn by the ubiquitin conjugating enzyme Cdc34 and SCF ubiquitin ligase complexes of the ubiquitin-proteasome system (UPS) to control the initiation of DNA replication. Here we demonstrate that the nutrient sensing kinases PKA and Sch9 phosphorylate S97 of Cdc34. S97 is conserved across species and restricted to the catalytic domain of Cdc34/Ubc7-like E2s. Cdc34-S97 phosphorylation is cell cycle regulated, elevated during active cell growth and division and decreased during cell cycle arrest. Cell growth and cell division are orchestrated to maintain cell size homeostasis over a wide range of nutrient conditions. Cells monitor changes in their environment through nutrient sensing protein kinases. Thus Cdc34 phosphorylation by PKA and Sch9 provides a direct tether between G1 cell division events and cell growth.
Assuntos
Domínio Catalítico , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Alimentos , Proteínas Quinases/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Ciclo Celular/fisiologia , Divisão Celular , Tamanho Celular , Humanos , Fosforilação , Especificidade da Espécie , Enzimas de Conjugação de Ubiquitina , Complexos Ubiquitina-Proteína Ligase/químicaRESUMO
The Cdc34 ubiquitin-conjugating enzyme plays a central role in progression of the cell cycle. Through analysis of the phenotype of a mutant missing a highly conserved sequence motif within the catalytic domain of Cdc34, we discovered previously unrecognized levels of regulation of the Ace2 transcription factor and the cyclin-dependent protein kinase inhibitor Sic1. In cells carrying the Cdc34(tm) mutation, which alters the conserved sequence, the cyclin-dependent protein kinase inhibitor Sic1, an SCF(Cdc4) substrate, has a shorter half-life, while the cyclin Cln1, an SCF(Grr1) substrate, has a longer half-life than in wild-type cells. Expression of the SIC1 gene cluster, which is regulated by Swi5 and Ace2 transcription factors, is induced in CDC34(tm) cells. Levels of Swi5, Ace2, and the SCF(Grr1) targets Cln1 and Cln2 are elevated in Cdc34(tm) cells, and loss of Grr1 causes an increase in Ace2 levels. Sic1 levels are similar in CDC34(tm) ace2Δ and wild-type cells, explaining a paradoxical increase in the steady-state level of Sic1 protein despite its reduced half-life. A screen for mutations that interact with CDC34(tm) uncovered novel regulators of Sic1, including genes encoding the polyubiquitin chain receptors Rad23 and Rpn10.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina/genética , Peptidil Dipeptidase A/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Enzima de Conversão de Angiotensina 2 , Ciclo Celular/genética , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Genes Sintéticos , Meia-Vida , Dados de Sequência Molecular , Peptidil Dipeptidase A/metabolismo , Poliubiquitina/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
BACKGROUND: The S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less triple mutant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no loop motif present in other E2s, supports growth. Whether tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown. RESULTS: tmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCFCdc4. Phosphorylation of C-terminal serines in tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, tmCDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in tmCDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure. CONCLUSIONS: The catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.
RESUMO
The pro-inflammatory cytokines, Tumor Necrosis Factor-alpha (TNFalpha) and Interleukin-1 (IL-1) mediate the innate immune response. Dysregulation of the innate immune response contributes to the pathogenesis of cancer, arthritis, and congestive heart failure. TNFalpha- and IL-1-induced changes in gene expression are mediated by similar transcription factors; however, TNFalpha and IL-1 receptor knock-out mice differ in their sensitivities to a known initiator (lipopolysaccharide, LPS) of the innate immune response. The contrasting responses to LPS indicate that TNFalpha and IL-1 regulate different processes. A large-scale proteomic analysis of TNFalpha- and IL-1-induced responses was undertaken to identify processes uniquely regulated by TNFalpha and IL-1. When combined with genomic studies, our results indicate that TNFalpha, but not IL-1, mediates cell cycle arrest.
Assuntos
Genômica , Interleucina-1/farmacologia , Proteômica , Fator de Necrose Tumoral alfa/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Membranas Mitocondriais/efeitos dos fármacos , Proteínas/análise , Proteínas/genética , RNA Mensageiro/análise , Transcrição Gênica/efeitos dos fármacosRESUMO
The presence of the appropriate pheromone induces alpha and a cells of the yeast Saccharomyces cerevisiae to activate both changes in transcriptional expression and cell polarity that eventually lead to the mating of alpha and a cells to form a/alpha diploid cells. A third response after exposure to mating pheromone is a transient cell cycle arrest, allowing synchronization of the two cell types in G1 prior to cell fusion. At least in part, this cell cycle arrest requires the inactivation of Cln-kinase activity through transcriptional inactivation of the CLN1 and CLN2 genes, degradation of the Cln proteins and direct inhibition of Cln-kinase complexes. Here we report that GRR1, which encodes a substrate recognition subunit of SCF complexes, is critical for pheromone sensitivity and likely for this arrest. Loss of SCF(Grr1) function by deletion of the GRR1 gene causes pheromone resistance. However, deletion of CLN1 and CLN2 restores pheromone sensitivity to grr1Delta cells. Thus, rapid loss of Cln-kinase activity during mating may require coordinated inactivation of the Cln-kinase complexes, inactivation of CLN transcription and SCF(Grr1)-dependent Cln degradation.
Assuntos
Regulação Fúngica da Expressão Gênica , Feromônios/farmacologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Aminopeptidases , Ciclo Celular/efeitos dos fármacos , Ciclinas/genética , Ciclinas/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases , Endopeptidases , Proteínas F-Box , Mutação , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Proteínas Ligases SKP Culina F-Box/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Serina Proteases , Tripeptidil-Peptidase 1 , Ubiquitina-Proteína Ligases/genéticaRESUMO
Covalent modifications to histone proteins are well documented in the literature. Specific modification sites are correlated with chromatin structure and transcriptional activity. The histone code is very complex, and includes several types of covalent modifications such as acetylation, methylation, phosphorylation, and ubiquitination of at least 20 possible sites within the histone proteins. The final chromatin structure "read-out" is a result of the cooperation between these many sites of covalent modifications. Methylation and acetylation sites of histone H3 from many different species have been previously identified. However, a full post-translational modification status on histone H3 from mouse has not yet been reported. Here we demonstrate the use of high-accuracy matrix-assisted laser desorption/ionization time-of-flight and nanoelectrospray ionization tandem mass spectrometry to identify the methylation and acetylation sites of the mouse histone H3. In addition to the sites previously identified from other species, one unique methylation site, Lys-122, from mouse histone H3 was identified. The reported mass spectrometric method provides an efficient and sensitive way for analyzing post-translational modifications of histone proteins.
Assuntos
Histonas/química , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Histonas/metabolismo , Lisina/química , Lisina/metabolismo , Metilação , Camundongos , Dados de Sequência Molecular , Nanotecnologia/métodosRESUMO
Cells treated with ultraviolet (UV) radiation undergo cell cycle arrest at the S-phase and G1/S boundary, allowing DNA repair to occur. Several proteins such as replication protein A and DNA-dependent protein kinase have been suggested to be involved in UV-induced inhibition of DNA replication. However, the role of these proteins in inhibiting DNA replication remains unknown. Other proteins may play important roles in modulating functions of these proteins in response to UV-irradiation. To understand the broad range of proteins involved in this inhibition, we carried out a systematic study to identify specific proteins involved in UV-induced replication arrest using two-dimensional gel electrophoresis and mass spectrometry. Unique changes in protein expression level for 31 proteins were observed over a 24-hour time course, including calgizzarin, cyclophilin A, and macrophage migration inhibitory factor. The expression level changes of these proteins are dynamically correlated to DNA replication activity, suggesting involvement of these proteins in modulating DNA replication and repair activities. This proteomic approach provides opportunities to gain insights into the mechanism by which DNA replication is inhibited.
Assuntos
Proteoma/análise , Proteoma/efeitos da radiação , Proteômica/métodos , Raios Ultravioleta , Reparo do DNA , Replicação do DNA/efeitos da radiação , Bases de Dados de Proteínas , Eletroforese em Gel Bidimensional , Expressão Gênica/efeitos da radiação , Perfilação da Expressão Gênica , Células HeLa , Humanos , Modelos Biológicos , Proteoma/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de TempoRESUMO
BACKGROUND: Beta-2-microglobulin (beta(2)M) amyloidosis is a destructive articular disease affecting dialysis patients. The amyloid deposits contain both beta(2)M and beta(2)M altered with advanced glycation end products (AGE-beta(2)M). We have shown that beta(2)M increases the expression of matrix metalloproteinase-1, vascular cell adhesion molecule-1 and cyclooxygenase-2 in human synovial fibroblasts, while the effect of AGE-beta(2)M in this model is markedly reduced. Conversely, in human monocyte/macrophages, AGE-beta(2)M stimulates cytokine release whereas beta(2)M is less potent. METHODS: To understand why the two forms of beta(2)M produce variable responses in different cells, AGE-beta(2)M was labelled with the fluorochrome Cy5, and beta(2)M was labelled with the fluorochrome Texas Red (TR) and the uptake of 50 microg/ml of each was examined through live cell imaging at different time points using confocal microscopy. RESULTS: In human synovial fibroblasts, the AGE-beta(2)M-Cy5 could be seen in endosome-like structures inside cells by 45 min. After 3.5 h the distribution of endosome-like structures had become perinuclear in nature and the concentration of AGE-beta(2)M-Cy5 within these structures had increased. When a 20-fold excess of AGE-BSA was added to the synovial fibroblasts with the AGE-beta(2)M-Cy5, the endosome-like particles were not seen, suggesting competitive inhibition of uptake through an AGE-receptor. In contrast, beta(2)M-TR progressively concentrated along the surface of synovial fibroblasts with minimal cellular uptake indicated by faint endosome-like structures seen only after 8 h. Interestingly, in a different model, human and mouse monocyte/macrophages, the AGE-beta(2)M-Cy5 and beta(2)M-TR had similar patterns of distribution and kinetics of uptake. CONCLUSION: Our results suggest that beta(2)M and AGE-beta(2)M are endocytosed via different mechanisms in human synovial fibroblasts and monocytes/macrophages. These results may offer a potential explanation of differences observed in cell culture experiments.
Assuntos
Arginina/análogos & derivados , Fibroblastos/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Lisina/análogos & derivados , Macrófagos/metabolismo , Membrana Sinovial/patologia , Microglobulina beta-2/metabolismo , Arginina/farmacologia , Transporte Biológico , Células Cultivadas , Endocitose , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Humanos , Lisina/farmacologia , Macrófagos/patologia , Macrófagos/ultraestrutura , Microscopia Eletrônica , Monócitos/citologia , Monócitos/metabolismo , Monócitos/ultraestrutura , Osteoartrite/patologia , Osteoartrite/cirurgia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Membrana Sinovial/ultraestrutura , Microglobulina beta-2/isolamento & purificaçãoRESUMO
Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules coded by up to 13 genes in insects and 4 genes in mammals. In insects PGRPs activate antimicrobial pathways in the hemolymph and cells, or are peptidoglycan (PGN)-lytic amidases. In mammals one PGRP is an antibacterial neutrophil protein. We report that human PGRP-L is a Zn2+-dependent N-acetylmuramoyl-l-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and l-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ialpha, PGRP-Ibeta, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal delta1-343 deletion mutant) is reduced. The Zn2+ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys-419 (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas three other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-l-alanine amidase and this function is conserved in prokaryotes, insects, and mammals.
Assuntos
Proteínas de Transporte/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Sequência de Carboidratos , Proteínas de Transporte/química , Primers do DNA , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Zinco/metabolismoRESUMO
Human CD34(+) cells, highly enriched for hematopoietic stem and progenitors, and CD15(+) cells, more terminally differentiated myeloid cells in blood, represent distinct maturation/differentiation stages. A proteomic approach was used to identify proteins differentially present in these two populations from human cord blood. Cytosolic proteins were extracted and subjected to two-dimensional gel electrophoresis followed by mass spectrometry. On average, 460 protein spots on each gel were detected; 112 and 15 proteins, respectively, were found to be differentially expressed or post-translationally modified in CD34(+) and CD15(+) cells. This suggests that CD34(+) cells have a relatively larger proteome than mature CD15(+) myeloid cells and production of many stem/progenitor cell-associated proteins ceases or is dramatically down-regulated as the CD34(+) cells undergo differentiation. Of approximately 140 protein spots, 47 different proteins were positively identified by mass spectrometry and database search; these proteins belong to several functional categories, including cell signaling, transcription factors, cytoskeletal proteins, metabolism, protein folding, and vesicle trafficking. Multiple heat shock proteins and chaperones, as well as proteins important for intracellular trafficking, were predominantly present in CD34(+) cells. Most of the identified proteins in CD34(+) cells are expressed in germ cell tumors, as well as in embryonal carcinoma and neuroblastoma. Approximately eight novel proteins, whose functions are unknown, were identified. This study presents, for the first time, global cellular protein expression patterns in human CD34(+) and CD15(+) cells, which should help to better understand intracellular processes involved in myeloid differentiation and add insight into the functional capabilities of these distinct cell types.