RESUMO
Cerenkov free-electron lasers have primarily operated on the fundamental guided mode of the dielectric waveguide. Higher-order generation would allow short wavelength emission in a relatively large scale resonator. In comparison to the fundamental mode, we find that gain on higher-order modes can be significant in a planar geometry. This analysis is presented with a discussion of practical limits.
RESUMO
We present in this paper compelling evidence supporting the three-wave traveling-wave theory developed by Pierce fifty years ago. The transition in a Smith-Purcell free-electron laser from low, through moderate amplified spontaneous emission, to strong gain conditions was carefully controlled. Below threshold, the emitted far-infrared power exhibits oscillations with a cubic dependence on the electron beam current. Both characteristics are expected in a three-wave interaction yet, to date, have not been observed.
RESUMO
A theoretical analysis of Smith-Purcell radiation at very high energies is presented. The energy per unit frequency and solid angle is expressed in closed form as a function of the grating geometry, beam energy, and viewing angles. A certain choice of grating geometry is shown to optimize the output energy for a particular order of radiation. Scaling laws are derived for the energy emitted into all orders of radiation in the relativistic limit. It is shown that the total energy emitted into each order scales as the three-halves power of the beam voltage.
RESUMO
Temporal control of p27(Kip1) (p27) degradation imposes periodicity in its activity during cell cycle progression and its accumulation during cell cycle exit. Degradation of p27 is initiated by phosphorylation of p27 at Thr-187, which marks the protein for ubiquitination by SCF(Skp2) and subsequent proteolysis by the 26S proteasome. Here we show that the p27 ubiquitination activity in cell extracts depends on the presence of the ubiquitin-like protein Nedd8 and enzymes that catalyze Nedd8 conjugation to proteins. Moreover, we show that reconstitution of the p27 ubiquitination activity of recombinant SCF(Skp2) also requires Nedd8 conjugation pathway components. Inactivation of the Nedd8 conjugation pathway by a dominant negative mutant of the Nedd8-conjugating enzyme Nce1/Ubc12 blocks the ubiquitination and degradation of p27 in cell extracts. Consistent with a role in cell-cycle progression, Nedd8 is expressed in proliferating cells and is itself down-regulated upon cellular differentiation. These results suggest that the Nedd8 conjugation pathway may regulate the turnover of p27(Kip1), independently of p27 phosphorylation, and further establishes the identity of protein components involved in p27 ubiquitination. Finally, these findings provide a direct demonstration of a function for Nedd8 in a biological process.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Inibidores Enzimáticos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Supressoras de Tumor , Complexos Ubiquitina-Proteína Ligase , Ubiquitinas/metabolismo , Substituição de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Sítios de Ligação , Domínio Catalítico , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/metabolismo , Cisteína , Escherichia coli , Células HeLa , Humanos , Cinética , Ligases/metabolismo , Mutagênese Sítio-Dirigida , Proteína NEDD8 , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Serina , Ubiquitina-Proteína LigasesRESUMO
Regulation of NF-kappaB occurs through phosphorylation-dependent ubiquitination of IkappaBalpha, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkappaBalpha is carried out by a ubiquitin-ligase enzyme complex called SCF(beta(TrCP)). Here we show that Nedd8 modification of the Cul-1 component of SCF(beta(TrCP)) is important for function of SCF(beta(TrCP)) in ubiquitination of IkappaBalpha. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF(beta(TrCP)), phosphorylated IkappaBalpha and beta-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCF(beta(TrCP)) in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed betaTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkappaBalpha required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF(beta(TrCP)) containing a K720R mutant of Cul-1 only weakly supported IkappaBalpha ubiquitination compared to SCF(beta(TrCP)) containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF(beta(TrCP)). These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkappaBalpha.
Assuntos
Proteínas de Ciclo Celular , Proteínas Culina , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Helminto/metabolismo , Proteínas I-kappa B , Peptídeo Sintases/metabolismo , Proteínas de Saccharomyces cerevisiae , Transativadores , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Helminto/genética , Humanos , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Proteína NEDD8 , Fosforilação , Proteínas Ligases SKP Culina F-Box , Alinhamento de Sequência , Transfecção , beta Catenina , Proteínas Contendo Repetições de beta-TransducinaAssuntos
Acetiltransferases/análise , Acetiltransferases/metabolismo , Núcleo Celular/ultraestrutura , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Animais , Fracionamento Celular/métodos , Núcleo Celular/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Galinhas , Cromatina/metabolismo , Cromatina/ultraestrutura , Cromatografia Líquida de Alta Pressão/métodos , Eritrócitos/ultraestrutura , Células HeLa , Histona Acetiltransferases , Histonas/isolamento & purificação , Humanos , Nuclease do Micrococo , Nucleossomos/ultraestrutura , Especificidade por SubstratoRESUMO
The inhibition of glyoxalase I enzyme to increase cellular levels of methylglyoxal has been developed as a rationale for the production of anticancer agents. Synthesis of a peptidomimetic analog of the previously prepared potent glyoxalase inhibitor, S-(p-bromobenzyl)glutathione (PBBG), was accomplished by inserting a urea linkage, NH-CO-NH, to replace the gamma-glutamyl peptide bond. Thus, the target compound, gamma-(L-gamma-azaglutamyl)-S-(p-bromobenzyl)-L-cysteinylglycine 6, was a potent inhibitor of glyoxalase I with almost no loss of activity when compared to PBBG. However, unlike PBBG, 6 was completely resistant to enzymatic degradation by kidney homogenate or by purified gamma-glutamyltranspeptidase enzyme.
Assuntos
Compostos Aza/síntese química , Glutationa/análogos & derivados , Lactoilglutationa Liase/antagonistas & inibidores , Animais , Cromatografia em Camada Fina , Glutationa/síntese química , Glutationa/farmacologia , Rim/metabolismo , Camundongos , Modelos Químicos , Fatores de TempoRESUMO
Recently, we reported the identification of a 55-kDa polypeptide (p55) from Tetrahymena macronuclei as a catalytic subunit of a transcription-associated histone acetyltransferase (HAT A). Extensive homology between p55 and Gcn5p, a component of the SAGA and ADA transcriptional coactivator complexes in budding yeast, suggests an immediate link between the regulation of chromatin structure and transcriptional output. Here we report the characterization of a second transcription-associated HAT activity from Tetrahymena macronuclei. This novel activity is distinct from complexes containing p55 and putative ciliate SAGA and ADA components and shares several characteristics with NuA4 (for nucleosomal H2A/H4), a 1.8-MDa, Gcn5p-independent HAT complex recently described in yeast. A key feature of both the NuA4 and Tetrahymena activities is their acetylation site specificity for lysines 5, 8, 12, and 16 of H4 and lysines 5 and 9 of H2A in nucleosomal substrates, patterns that are distinct from those of known Gcn5p family members. Moreover, like NuA4, the Tetrahymena activity is capable of activating transcription from nucleosomal templates in vitro in an acetyl coenzyme A-dependent fashion. Unlike NuA4, however, sucrose gradient analyses of the ciliate enzyme, following sequential denaturation and renaturation, estimate the molecular size of the catalytically active subunit to be approximately 80 kDa, consistent with the notion that a single polypeptide or a stable subcomplex is sufficient for this H2A/H4 nucleosomal HAT activity. Together, these data document the importance of this novel HAT activity for transcriptional activation from chromatin templates and suggest that a second catalytic HAT subunit, in addition to p55/Gcn5p, is conserved between yeast and Tetrahymena.
Assuntos
Acetiltransferases/metabolismo , Nucleossomos/enzimologia , Proteínas de Saccharomyces cerevisiae , Tetrahymena thermophila/enzimologia , Acetilação , Acetiltransferases/isolamento & purificação , Animais , Catálise , Cromatina , Cromatografia Líquida de Alta Pressão , Células HeLa , Histona Acetiltransferases , Humanos , Moldes Genéticos , Transcrição Gênica , LevedurasAssuntos
Cromatina/fisiologia , Replicação do DNA , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Transdução de Sinais , Transcrição Gênica , Acetilação , Acetiltransferases/metabolismo , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , DNA/química , DNA/genética , Histona Acetiltransferases , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , FosforilaçãoRESUMO
The transcriptional adaptor protein Gcn5 has been identified as a nuclear histone acetyltransferase (HAT). Although recombinant yeast Gcn5 efficiently acetylates free histones, it fails to acetylate histones contained in nucleosomes, indicating that additional components are required for acetylation of chromosomal histones. We report here that Gcn5 functions as a catalytic subunit in two high-molecular-mass native HAT complexes, with apparent molecular masses of 0.8 and 1.8 megadalton (MD), respectively, which acetylate nucleosomal histones. Both the 0.8- and 1.8-MD Gcn5-containing complexes cofractionate with Ada2 and are lost in gcn5delta, ada2delta, or ada3delta yeast strains, illustrating that these HAT complexes are bona fide native Ada-transcriptional adaptor complexes. Importantly, the 1.8-MD adaptor/HAT complex also contains Spt gene products that are linked to TATA-binding protein (TBP) function. This complex is lost in spt20/ada5delta and spt7delta strains and Spt3, Spt7, Spt20/Ada5, Ada2, and Gcn5 all copurify with this nucleosomal HAT complex. Therefore, the 1.8-MD adaptor/HAT complex illustrates an interaction between Ada and Spt gene products and confirms the existence of a complex containing the TBP group of Spt proteins as demonstrated by genetic and biochemical studies. We have named this novel transcription regulatory complex SAGA (Spt-Ada-Gcn5-Acetyltransferase). The function of Gcn5 as a histone acetyltransferase within the Ada and SAGA adaptor complexes indicates the importance of histone acetylation during steps in transcription activation mediated by interactions with transcription activators and general transcription factors (i.e., TBP).
Assuntos
Acetiltransferases/metabolismo , Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Complexos Multienzimáticos/metabolismo , Nucleossomos/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Acetilação , Acetiltransferases/genética , Catálise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/genética , Histona Acetiltransferases , Complexos Multienzimáticos/genética , Proteínas Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Yeast and human ADA2 and GCN5 (y- and hADA2 and y- and hGCN5, respectively) have been shown to potentiate transcription in vivo and may function as adaptors to bridge physical interactions between DNA-bound activators and the basal transcriptional machinery. Recently it was shown that yGCN5 is a histone acetyltransferase (HAT), suggesting a link between enzymatic modification of nucleosomes and transcriptional activation. In this report, we demonstrate that hGCN5 is also an HAT and has the same substrate specificity as yGCN5. Since hGCN5 does not complement functional defects caused by deletion of yGCN5, we constructed a series of hGCN5-yGCN5 chimeras to identify human regions capable of activity in yeast. Interestingly, only the putative HAT domain of hGCN5, when fused to the remainder of yGCN5, complemented gcn5- cells for growth and transcriptional activation. Moreover, an amino acid substitution mutation within the HAT domain reduced both HAT activity in vitro and transcription in vivo. These findings directly link enzymatic histone acetylation and transcriptional activation and show evolutionary conservation of this potentially crucial pathway in gene regulation.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Ativação Transcricional/fisiologia , Acetilação , Proteínas Fúngicas/genética , Teste de Complementação Genética , Glutationa Transferase/genética , Proteína Vmw65 do Vírus do Herpes Simples/genética , Histona Acetiltransferases , Histonas/metabolismo , Humanos , Mutação , Proteínas Quinases/genética , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Especificidade por Substrato , Fatores de Transcrição/metabolismoRESUMO
The transcription initiation factor TFIID is a multimeric protein complex composed of TATA box-binding protein (TBP) and many TBP-associated factors (TAF(II)s). TAF(II)s are important cofactors that mediate activated transcription by providing interaction sites for distinct activators. Here, we present evidence that human TAF(II)250 and its homologs in Drosophila and yeast have histone acetyltransferase (HAT) activity in vitro. HAT activity maps to the central, most conserved portion of dTAF(II)230 and yTAF(II)130. The HAT activity of dTAF(II)230 resembles that of yeast and human GCN5 in that it is specific for histones H3 and H4 in vitro. Our findings suggest that targeted histone acetylation at specific promoters by TAF(II)250 may be involved in mechanisms by which TFIID gains access to transcriptionally repressed chromatin.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Drosophila melanogaster , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica , Células HeLa , Histona Acetiltransferases , Humanos , Proteínas de Insetos/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Peptídeos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Recombinantes , Saccharomyces cerevisiae , Deleção de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato , Fator de Transcrição TFIID , Transcrição GênicaRESUMO
The yeast transcriptional adaptor, Gcn5p, is a catalytic subunit of a nuclear (type A) histone acetyltransferase linking histone acetylation to gene activation. Here we report that Gcn5p acetylates histones H3 and H4 non-randomly at specific lysines in the amino-terminal domains. Lysine 14 of H3 and lysines 8 and 16 of H4 are highly preferred acetylation sites for Gcn5p. We also demonstrate that lysine 9 is the preferred position of acetylation in newly synthesized yeast H3 in vivo. This finding, along with the fact that lysines 5 and 12 in H4 are predominant acetylation sites during chromatin assembly of many organisms, indicates that Gcn5p acetylates a distinct set of lysines that do not overlap with those sites characteristically used by type B histone acetyltransferases for histone deposition and chromatin assembly.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ligação a DNA , Proteínas Fúngicas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Transcrição Gênica , Acetilação , Sequência de Aminoácidos , Animais , Bovinos , Galinhas , Histona Acetiltransferases , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Especificidade por Substrato , Tetrahymena thermophila/metabolismoRESUMO
Post-translational acetylation of the core histone amino-terminal tails has long been associated with both chromatin assembly and the regulation of gene expression. The recent identification and cloning of histone acetyltransferase genes represents a significant breakthrough in our understanding of how specific acetylation states are established. Ongoing characterization of these enzymes and their molecular cohorts supports a direct role for acetylation in a signaling pathway that modulates chromatin structure to create new patterns of transcription.
Assuntos
Acetiltransferases/fisiologia , Cromatina/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Saccharomyces cerevisiae , Acetilação , Sequência de Aminoácidos , Histona Acetiltransferases , Histonas/metabolismo , Dados de Sequência Molecular , Ativação TranscricionalRESUMO
We report the cloning of a transcription-associated histone acetyltransferase type A(HAT A). This Tetrahymena enzyme is strikingly homologous to the yeast protein Gcn5, a putative transcriptional adaptor, and we demonstrate that recombinant Gcn5p possesses HAT activity. Both the ciliate enzyme and Gcn5p contain potential active site residues found in other acetyltransferases and a highly conserved bromodomain. The presence of this domain in nuclear A-type HATs, but not in cytoplasmic B-type HATs, suggests a mechanism whereby HAT A is directed to chromatin to facilitate transcriptional activation. These findings shed light on the biochemical function of the evolutionarily conserved Gcn5p-Ada complex, directly linking histone acetylation to gene activation, and indicate that histone acetylation is a targeted phenomenon.
Assuntos
Acetiltransferases/genética , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Tetrahymena thermophila/genética , Acetilação , Acetiltransferases/metabolismo , Animais , Sequência de Bases , Cromatina/genética , Clonagem Molecular , Sequência Conservada , Regulação Enzimológica da Expressão Gênica/fisiologia , Genes de Protozoários/fisiologia , Histona Acetiltransferases , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Ativação Transcricional , Leveduras/genéticaRESUMO
Macronuclei of the ciliated protozoan Tetrahymena thermophila possess a histone acetyltransferase activity closely associated with transcription-related histone acetylation. Nothing definitive is known concerning the polypeptide composition of this activity in Tetrahymena or any comparable activity from any cellular source. An acetyltransferase activity gel assay was developed which identifies a catalytically active subunit of this enzyme in Tetrahymena. This activity gel assay detects a single polypeptide of 55 kDa (p55) in crude macronuclear extracts, as well as in column-purified fractions, which incorporates [3H]acetate from [3H]acetyl-CoA into core histone substrates polymerized directly into SDS polyacrylamide gels. p55 copurifies precisely with acetyltransferase activity through all chromatographic steps examined, including reverse-phase HPLC. Gel-filtration chromatography of this activity indicates a molecular mass of 220 kDa, suggesting that the native enzyme may consist of four identical subunits of 55 kDa. Furthermore, p55 is tightly associated with di- and greater polynucleosomes and therefore may be defined as a component of histone acetyltransferase type A--i.e., chromatin associated.
Assuntos
Acetiltransferases/metabolismo , Núcleo Celular/enzimologia , Cromatina/enzimologia , Proteínas de Saccharomyces cerevisiae , Tetrahymena thermophila/enzimologia , Acetiltransferases/análise , Acetiltransferases/isolamento & purificação , Animais , Fracionamento Celular , Cromatografia , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Durapatita , Eletroforese em Gel de Poliacrilamida , Histona Acetiltransferases , Cinética , Substâncias Macromoleculares , Peso MolecularRESUMO
Two new peptides, a diketopiperazine of N-methyltyrosine (1) and a tetrapeptide containing N-methyltyrosine (2), were isolated from an actinomycete strain Streptomyces griseus. These compounds inhibit the enzyme calpain in the micromolar range and were characterized on the basis of spectroscopic analysis, amino acid analysis and sequencing. The structure of the tetrapeptide N-methyltyrosyl-N-methyltyrosyl-leucyl-alanine (2), was also confirmed by total synthesis.
Assuntos
Calpaína/antagonistas & inibidores , Oligopeptídeos/isolamento & purificação , Piperazinas/isolamento & purificação , Streptomyces griseus/metabolismo , Tirosina/análogos & derivados , Inibidores Enzimáticos/química , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Espectroscopia de Ressonância Magnética , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Tirosina/química , Tirosina/isolamento & purificação , Tirosina/farmacologiaRESUMO
A novel radiometric assay was developed for human fibroblast collagenase (matrix metalloproteinase-1, MMP-1), stromelysin (MMP-3), and their recombinant catalytic domains. Using this assay we were able to compare the native MMPs with the respective catalytic domains in terms of inhibitor affinities and peptide hydrolysis. The assay works on the same principle as an assay developed for carboxypeptidase (Rossier et al., Anal. Biochem. 1989, 178, 27-31) and is based on a synthetic peptide substrate, [1-benzoyl-14C]benzoyl-Pro-Leu-Ala-Leu-Trp- NH(CH2)4N(CH3)2(bnzPLALW-NX). The generation of product is measured by selective solvent extraction of radioactive product directly into scintillation cocktail; the entire assay, including the radioactivity measurement, is completed in a single 1-ml tube (96-well format) without removal or transfer of phases. Results of steady-state measurements demonstrated that peptide hydrolysis follows Michaelis-Menten kinetics with the fibroblast MMPs and their C-terminal deleted forms. The kinetic constants for hydrolysis of bnz-PLALW-NX, and for inhibition by actinonin, a natural peptide-hydroxamate, are essentially the same for the native and the C-terminally deleted MMPs.
Assuntos
Colagenases/metabolismo , Metaloendopeptidases/metabolismo , Sítios de Ligação , Catálise , Humanos , Hidrólise , Ácidos Hidroxâmicos/farmacologia , Cinética , Metaloproteinase 1 da Matriz , Metaloproteinase 3 da Matriz , Inibidores de Metaloproteinases de Matriz , Peptídeos/metabolismo , Radiometria , Proteínas Recombinantes/metabolismoRESUMO
WIN 64821, a nonpeptide neurokinin antagonist, was isolated from a strain of Aspergillus sp., SC319. The compound was produced in different fermentation media with greatest yields observed when the culture was grown in a synthetic medium supplemented with L-tryptophan and L-phenylalanine. After 6 days fermentation, yields greater than 600 mg/liter were obtained. Two analogs of WIN 64821 were also identified in the culture extracts and subsequently tested for biological activity. WIN 64821 was the most potent compound isolated from this culture and exhibited activity as a substance P-binding inhibitor with submicromolar potency against the human neurokinin 1 receptor.