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1.
Methods ; 65(1): 11-7, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-23688935

RESUMO

Since the introduction of "soft" ionization techniques, the role of mass spectrometry (MS) in the field of structural biology has increasingly expanded. With the incorporation of volatile buffers as electrospray ionization (ESI) solvents, non-covalent protein complexes could be efficiently transferred to the gas phase for mass analysis. While native MS has not become a technique used for standard characterization of therapeutic proteins in an industrial setting, it is increasingly used to probe the structural heterogeneity of these complex biomolecules. Here, we describe a detailed sample protocol for the analysis of monoclonal antibodies (mAbs) by native MS and highlight some recent applications of native MS in the analysis of intact mAbs and mAb-based therapeutics.


Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Espectrometria de Massas por Ionização por Electrospray , Animais , Anticorpos Monoclonais/uso terapêutico , Glicosídeo Hidrolases/química , Glicosilação , Humanos , Peso Molecular , Processamento de Proteína Pós-Traducional
2.
MAbs ; 6(1): 197-203, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24351421

RESUMO

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to batch characterization.


Assuntos
Espectrometria de Massas/métodos , Anticorpos de Cadeia Única/química , Células HEK293 , Humanos
3.
Structure ; 22(2): 281-90, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24373769

RESUMO

The protease-chaperone DegP undergoes secondary through quaternary structural changes, regulating function and preventing indiscriminate proteolysis. Several structures of DegP oligomers have been observed, including the resting state 6-mer and the 12-mer and 24-mer active states. However, the precise events of the transition between the resting and active states still need to be elucidated. We used native mass spectrometry to demonstrate that binding of multiple substrate-mimicking peptide ligands to the DegP resting state occurs prior to the transition to an active conformation. This transition occurred at a 6-mer occupancy of 40% for each peptide ligand. We observed ligand-specific 9-mer formation with a maximum load of 9 peptides, whereas other substrates led to 12-mers accommodating 24 peptides. Based on these data, we present a model for the initial steps of substrate-induced transitions from the resting to active states of DegP.


Assuntos
Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Proteínas Periplásmicas/química , Serina Endopeptidases/química , Sequência de Aminoácidos , Ligantes , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Dobramento de Proteína , Estrutura Quaternária de Proteína , Especificidade por Substrato
4.
PLoS One ; 8(5): e64040, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23737964

RESUMO

BACKGROUND: The 16 kDa heat shock protein (HSP) is an immuno-dominant antigen, used in diagnosis of infectious Mycobacterium tuberculosis (M.tb.) causing tuberculosis (TB). Its use in serum-based diagnostics is limited, but for the direct identification of M.tb. bacteria in sputum or cultures it may represent a useful tool. Recently, a broad set of twelve 16 kDa specific heavy chain llama antibodies (VHH) has been isolated, and their utility for diagnostic applications was explored. METHODOLOGY/PRINCIPAL FINDINGS: To identify the epitopes recognized by the nine (randomly selected from a set of twelve 16 kDa specific VHH antibodies) distinct VHH antibodies, 14 overlapping linear epitopes (each 20 amino acid long) were characterized using direct and sandwich ELISA techniques. Seven out of 14 epitopes were recognized by 8 out of 9 VHH antibodies. The two highest affinity binders B-F10 and A-23 were found to bind distinct epitopes. Sandwich ELISA and SPR experiments showed that only B-F10 was suitable as secondary antibody with both B-F10 and A-23 as anchoring antibodies. To explain this behavior, the epitopes were matched to the putative 3D structure model. Electrospray ionization time-of-flight mass spectrometry and size exclusion chromatography were used to determine the higher order conformation. A homodimer model best explained the differential immunological reactivity of A-23 and B-F10 against heat-treated M.tb. lysates. CONCLUSIONS/SIGNIFICANCE: The concentrations of secreted antigens of M.tb. in sputum are too low for immunological detection and existing kits are only used for identifying M.tb. in cultures. Here we describe how specific combinations of VHH domains could be used to detect the intracellular HSP antigen. Linked to methods of pre-concentrating M.tb. cells prior to lysis, HSP detection may enable the development of protein-based diagnostics of sputum samples and earlier diagnosis of diseases.


Assuntos
Proteínas de Bactérias/metabolismo , Camelídeos Americanos , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Cadeias Pesadas de Imunoglobulinas/imunologia , Mycobacterium tuberculosis/isolamento & purificação , Multimerização Proteica , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Mapeamento de Epitopos , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/imunologia , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Estrutura Quaternária de Proteína , Escarro/microbiologia , Tuberculose/diagnóstico
5.
Chem Commun (Camb) ; 49(6): 538-48, 2013 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-23183499

RESUMO

Monoclonal antibodies (mAbs) are important therapeutics, targeting a variety of diseases ranging from cancers to neurodegenerative disorders. In developmental stages and prior to clinical use, these molecules require thorough structural characterisation, but their large size and heterogeneity present challenges for most analytical techniques. Over the past 20 years, mass spectrometry (MS) has transformed from a tool for small molecule analysis to a technique that can be used to study large intact proteins and non-covalent protein complexes. Here, we review several MS-based techniques that have emerged for the analysis of intact mAbs and discuss the prospects of using these technologies for the analysis of biopharmaceuticals.


Assuntos
Anticorpos Monoclonais/química , Espectrometria de Massas , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Complexo Antígeno-Anticorpo/química , Medição da Troca de Deutério , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
6.
Angew Chem Int Ed Engl ; 51(52): 12992-6, 2012 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-23172610

RESUMO

Antibody profiling: native mass spectrometry analysis of intact antibodies can be achieved with improved speed, sensitivity, and mass resolution by using a modified orbitrap instrument. Complex mixtures of monoclonal antibodies can be resolved and their glycan "fingerprints" can be profiled. Noncovalent interactions are maintained, thus allowing antibody-antigen binding to be measured.


Assuntos
Anticorpos Monoclonais/química , Espectrometria de Massas , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Antígenos/imunologia , Glicosilação , Células HEK293 , Humanos , Interleucina-6/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia
7.
Anal Chem ; 84(16): 7227-32, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22882109

RESUMO

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.


Assuntos
Anticorpos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Anticorpos/isolamento & purificação , Células CHO , Cromatografia por Troca Iônica , Cricetinae , Cricetulus , Imunoglobulina G/análise , Imunoglobulina G/isolamento & purificação
8.
Mol Cell Proteomics ; 7(3): 519-33, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18056057

RESUMO

Defective mobilization of Ca2+ by cardiomyocytes can lead to cardiac insufficiency, but the causative mechanisms leading to congestive heart failure (HF) remain unclear. In the present study we performed exhaustive global proteomics surveys of cardiac ventricle isolated from a mouse model of cardiomyopathy overexpressing a phospholamban mutant, R9C (PLN-R9C), and exhibiting impaired Ca2+ handling and death at 24 weeks and compared them with normal control littermates. The relative expression patterns of 6190 high confidence proteins were monitored by shotgun tandem mass spectrometry at 8, 16, and 24 weeks of disease progression. Significant differential abundance of 593 proteins was detected. These proteins mapped to select biological pathways such as endoplasmic reticulum stress response, cytoskeletal remodeling, and apoptosis and included known biomarkers of HF (e.g. brain natriuretic peptide/atrial natriuretic factor and angiotensin-converting enzyme) and other indicators of presymptomatic functional impairment. These altered proteomic profiles were concordant with cognate mRNA patterns recorded in parallel using high density mRNA microarrays, and top candidates were validated by RT-PCR and Western blotting. Mapping of our highest ranked proteins against a human diseased explant and to available data sets indicated that many of these proteins could serve as markers of disease. Indeed we showed that several of these proteins are detectable in mouse and human plasma and display differential abundance in the plasma of diseased mice and affected patients. These results offer a systems-wide perspective of the dynamic maladaptions associated with impaired Ca2+ homeostasis that perturb myocyte function and ultimately converge to cause HF.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Cardiomiopatia Dilatada/metabolismo , Mutação/genética , Análise Serial de Proteínas , Proteômica/métodos , Estresse Fisiológico/metabolismo , Animais , Biomarcadores/sangue , Cardiomiopatia Dilatada/sangue , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/fisiopatologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca , Hemodinâmica , Humanos , Masculino , Redes e Vias Metabólicas , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo , Ultrassonografia
9.
Chemistry ; 13(34): 9637-46, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17847145

RESUMO

Five diamines with thiophene-based bridges--(E)-1,2-bis{5-[bis(4-butoxyphenyl)amino]-2-thienyl}ethylene (1), 5,5'-bis[bis(4-methoxyphenyl)amino]-2,2'-bithiophene (2), 2,6-bis[bis(4-butoxyphenyl)amino]dithieno[3,2-b:2',3'-d]thiophene (3), N-(4-tert-butylphenyl)-2,6-bis[bis(4-methoxyphenyl)amino]dithieno[3,2-b:2',3'-d]pyrrole (4 a) and N-tert-butyl-2,6-bis[bis(4-methoxyphenyl)amino]dithieno[3,2-b:2',3'-d]pyrrole (4 b)--have been synthesised. The syntheses make use of the palladium(0)-catalysed coupling of brominated thiophene species with diarylamines, in some cases accelerated by microwave irradiation. The molecules all undergo facile oxidation, 4 b being the most readily oxidised at about -0.4 V versus ferrocenium/ferrocene, and solutions of the corresponding radical cations were generated by addition of tris(4-bromophenyl)aminium hexachloroantimonate to the neutral species. The near-IR spectra of the radical cations show absorptions characteristic of symmetrical delocalised species (that is, class III mixed-valence species); analysis of these absorptions in the framework of Hush theory indicates strong coupling between the two amine redox centres, stronger than that observed in species with phenylene-based bridging groups of comparable length. The strong coupling can be attributed to high-lying orbitals of the thiophene-based bridging units. ESR spectroscopy indicates that the coupling constant to the amino nitrogen atoms is somewhat reduced relative to that in a stilbene-bridged analogue. The neutral species and the corresponding radical cations have been studied with the aid of density functional theory and time-dependent density functional theory. The DFT-calculated ESR parameters are in good agreement with experiment, while calculated spin densities suggest increased bridge character to the oxidation in these species relative to that in comparable species with phenylene-based bridges.

10.
Int J Cancer ; 120(9): 1914-21, 2007 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-17236199

RESUMO

Esophageal cancer is a particularly aggressive tumor with poor prognosis, however, our current knowledge of the genes and pathways involved in tumorigenesis of the esophagus are limited. To obtain insight into the molecular processes underlying tumorigenesis of the esophagus, we have used cDNA microarrays to compare the gene expression profiles of 128 tissue samples representing the major histological subtypes of esophageal cancer (squamous cell carcinoma and adenocarcinoma (ADC)) as well as Barrett's esophagus (BE), the precursor lesion to ADC, and normal esophageal epithelium. Linear discriminant analysis and unsupervised hierarchical clustering show the separation of samples into 4 distinct groups consistent with their histological subtype. Differentially expressed genes were identified between each of the tissue types. Comparison of gene ontologies and gene expression profiles identified gene profiles specific to esophageal cancer, as well as BE. "Esophageal cancer clusters," representing proliferation, immune response, and extracellular matrix genes were identified, as well as digestion, hydrolase, and transcription factor clusters specific to the columnar phenotype observed during BE and esophageal ADC. These clusters provide valuable insight into the molecular and functional differences between normal esophageal epithelium, BE, and the 2 histologically distinct forms of esophageal cancers. Our thorough, unbiased analysis provides a rich source of data for further studies into the molecular basis of tumorigenesis of the esophagus, as well as identification of potential biomarkers for early detection of progression.


Assuntos
Adenocarcinoma/genética , Esôfago de Barrett/genética , Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Análise por Conglomerados , Humanos , Análise de Sequência com Séries de Oligonucleotídeos
11.
Nature ; 440(7084): 637-43, 2006 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-16554755

RESUMO

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.


Assuntos
Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Evolução Biológica , Sequência Conservada , Espectrometria de Massas , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteoma/química , Proteômica , Proteínas de Saccharomyces cerevisiae/química
12.
Cell ; 123(3): 507-19, 2005 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-16269340

RESUMO

We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.


Assuntos
Epistasia Genética , Perfilação da Expressão Gênica , Mapeamento de Interação de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Análise por Conglomerados , Biologia Computacional , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Glicosilação , Proteínas de Membrana/genética , Mutação , Transporte Proteico/genética , Receptores de Peptídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
13.
Cell ; 123(4): 593-605, 2005 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-16286008

RESUMO

The yeast histone deacetylase Rpd3 can be recruited to promoters to repress transcription initiation. Biochemical, genetic, and gene-expression analyses show that Rpd3 exists in two distinct complexes. The smaller complex, Rpd3C(S), shares Sin3 and Ume1 with Rpd3C(L) but contains the unique subunits Rco1 and Eaf3. Rpd3C(S) mutants exhibit phenotypes remarkably similar to those of Set2, a histone methyltransferase associated with elongating RNA polymerase II. Chromatin immunoprecipitation and biochemical experiments indicate that the chromodomain of Eaf3 recruits Rpd3C(S) to nucleosomes methylated by Set2 on histone H3 lysine 36, leading to deacetylation of transcribed regions. This pathway apparently acts to negatively regulate transcription because deleting the genes for Set2 or Rpd3C(S) bypasses the requirement for the positive elongation factor Bur1/Bur2.


Assuntos
Histona Desacetilases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Análise por Conglomerados , Quinases Ciclina-Dependentes/genética , Regulação para Baixo/genética , Epistasia Genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Histona Desacetilases/genética , Metilação , Metiltransferases/genética , Dados de Sequência Molecular , Mutação/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ligação Proteica , RNA Polimerase II/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2 , Sirtuínas/genética , Sirtuínas/metabolismo , Supressão Genética , Fatores de Transcrição/genética
14.
J Med Chem ; 48(10): 3481-91, 2005 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-15887957

RESUMO

1H-Imidazo-[4,5-c]quinolines were prepared while investigating novel nucleoside analogues as potential antiviral agents. While these compounds showed no direct antiviral activity when tested in a number of cell culture systems, some demonstrated potent inhibition of virus lesion development in an intravaginal guinea pig herpes simplex virus-2 assay. We have determined that the in vivo antiviral activity can be attributed to the ability of these molecules to induce the production of cytokines, especially interferon (IFN), in this model. Subsequently, we found that the compounds also induce in vitro production of IFN in human peripheral blood mononuclear cells (hPBMCs). The in vitro results reported herein and the in vivo results reported previously led to the discovery of imiquimod, 26, which was developed as a topical agent and has been approved for the treatment of genital warts, actinic keratosis, and superficial basal cell carcinoma.


Assuntos
Aminoquinolinas/síntese química , Antivirais/síntese química , Imidazóis/síntese química , Indutores de Interferon/síntese química , Interferons/biossíntese , Quinolinas/síntese química , Aminoquinolinas/química , Aminoquinolinas/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Linhagem Celular Tumoral , Células Cultivadas , Efeito Citopatogênico Viral/efeitos dos fármacos , Cobaias , Herpesvirus Humano 2/efeitos dos fármacos , Humanos , Imidazóis/química , Imidazóis/farmacologia , Imiquimode , Indutores de Interferon/química , Indutores de Interferon/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Quinolinas/química , Quinolinas/farmacologia , Relação Estrutura-Atividade
15.
Mol Cell Biol ; 25(3): 1162-72, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15657441

RESUMO

The SAGA complex is a multisubunit protein complex involved in transcriptional regulation in Saccharomyces cerevisiae. SAGA combines proteins involved in interactions with DNA-bound activators and TATA-binding protein (TBP), as well as enzymes for histone acetylation (Gcn5) and histone deubiquitylation (Ubp8). We recently showed that H2B ubiquitylation and Ubp8-mediated deubiquitylation are both required for transcriptional activation. For this study, we investigated the interaction of Ubp8 with SAGA. Using mutagenesis, we identified a putative zinc (Zn) binding domain within Ubp8 as being critical for the association with SAGA. The Zn binding domain is required for H2B deubiquitylation and for growth on media requiring Ubp8's function in gene activation. Furthermore, we identified an 11-kDa subunit of SAGA, Sgf11, and showed that it is required for the Ubp8 association with SAGA and for H2B deubiquitylation. Different approaches indicated that the functions of Ubp8 and Sgf11 are related and separable from those of other components of SAGA. In particular, the profiles of Ubp8 and Sgf11 deletions were remarkably similar in microarray analyses and synthetic genetic interactions and were distinct from those of the Spt3 and Spt8 subunits of SAGA, which are involved in TBP regulation. These data indicate that Ubp8 and Sgf11 likely represent a new functional module within SAGA that is involved in gene regulation through H2B deubiquitylation.


Assuntos
Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismo , Sequência de Aminoácidos , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Histona Acetiltransferases , Análise em Microsséries , Dados de Sequência Molecular , Mutação/genética , Proteínas Quinases/metabolismo , Ativação Transcricional , Zinco/metabolismo
16.
J Phys Chem A ; 109(41): 9346-52, 2005 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16833276

RESUMO

The electron-donor abilities of ten aminophenyl systems and an additional aminothienyl system are compared using density functional theory calculations. The systems studied here include those with amine nitrogen atoms bearing alkyl or aryl groups and those with amine nitrogen atoms as part of a heterocycle. Their abilities to act as donors in electron-transfer processes are assessed from calculated vertical ionization potentials for the aminobenzenes, which are in good agreement with available experimental data. Their abilities to act as intramolecular pi-electron donors in conjugated systems are inferred from the bond lengths and charge densities calculated for the corresponding 4-aminobenzaldehydes and 4-aminobenzonitriles. The computed (13)C NMR chemical shifts for the 4-aminobenzaldehydes and 4-aminobenzonitriles are in good agreement with published and new experimental data. The chemical shifts correlate well with the computed charge densities and can, to some extent, be used as an experimental probe of pi-donor strength. We find that the electron-transfer-donor strengths do not correlate well with pi-donor strengths: these differences can largely be attributed to steric effects.

17.
Proc Natl Acad Sci U S A ; 101(37): 13513-8, 2004 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-15353583

RESUMO

NuA4, the only essential histone acetyltransferase complex in Saccharomyces cerevisiae, acetylates the N-terminal tails of histones H4 and H2A. Affinity purification of NuA4 revealed the presence of three previously undescribed subunits, Vid21/Eaf1/Ydr359c, Swc4/Eaf2/Ygr002c, and Eaf7/Ynl136w. Experimental analyses revealed at least two functionally distinct sets of polypeptides in NuA4: (i) Vid21 and Yng2, and (ii) Eaf5 and Eaf7. Vid21 and Yng2 are required for bulk histone H4 acetylation and are functionally linked to the histone H2A variant Htz1 and the Swr1 ATPase complex (SWR-C) that assembles Htz1 into chromatin, whereas Eaf5 and Eaf7 have a different, as yet undefined, role. Mutations in Htz1, the SWR-C, and NuA4 cause defects in chromosome segregation that are consistent with genetic interactions we have observed between the genes encoding these proteins and genes encoding kinetochore components. Because SWR-C-dependent recruitment of Htz1 occurs in both transcribed and centromeric regions, a NuA4/SWR-C/Htz1 pathway may regulate both transcription and centromere function in S. cerevisiae.


Assuntos
Acetiltransferases/metabolismo , Adenosina Trifosfatases/metabolismo , Instabilidade Cromossômica , Histonas/genética , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Acetiltransferases/isolamento & purificação , Adenosina Trifosfatases/genética , Montagem e Desmontagem da Cromatina , Instabilidade Cromossômica/genética , Cromossomos Fúngicos/metabolismo , Histona Acetiltransferases , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
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