RESUMO
Pulmonary arterial hypertension (PAH) is characterized by obliterative vascular remodeling of the small pulmonary arteries (PA) and progressive increase in pulmonary vascular resistance (PVR) leading to right ventricular (RV) failure. Although several drugs are approved for the treatment of PAH, mortality remains high. Accumulating evidence supports a pathological function of integrins in vessel remodeling, which are gaining renewed interest as drug targets. However, their role in PAH remains largely unexplored. We found that the arginine-glycine-aspartate (RGD)-binding integrin α5ß1 is upregulated in PA endothelial cells (PAEC) and PA smooth muscle cells (PASMC) from PAH patients and remodeled PAs from animal models. Blockade of the integrin α5ß1 or depletion of the α5 subunit resulted in mitotic defects and inhibition of the pro-proliferative and apoptosis-resistant phenotype of PAH cells. Using a novel small molecule integrin inhibitor and neutralizing antibodies, we demonstrated that α5ß1 integrin blockade attenuates pulmonary vascular remodeling and improves hemodynamics and RV function in multiple preclinical models. Our results provide converging evidence to consider α5ß1 integrin inhibition as a promising therapy for pulmonary hypertension. One sentence summary: The α5ß1 integrin plays a crucial role in pulmonary vascular remodeling.
RESUMO
Collagen superfamily of proteins is a major component of the extracellular matrix. Defects in collagens underlie the cause of nearly 40 human genetic diseases in millions of people worldwide. Pathogenesis typically involves genetic alterations of the triple helix, a hallmark structural feature that bestows exceptional mechanical resistance to tensile forces and a capacity to bind a plethora of macromolecules. Yet, there is a paramount knowledge gap in understanding the functionality of distinct sites along the triple helix. Here, we present a recombinant technique to produce triple helical fragments for functional studies. The experimental strategy utilizes the unique capacity of the NC2 heterotrimerization domain of collagen IX to drive three α-chain selection and registering the triple helix stagger. For proof of principle, we produced and characterized long triple helical fragments of collagen IV that were expressed in a mammalian system. The heterotrimeric fragments encompassed the CB3 trimeric peptide of collagen IV, which harbors the binding motifs for α1ß1 and α2ß1 integrins. Fragments were characterized and shown to have a stable triple helix, post-translational modifications, and high affinity and specific binding of integrins. The NC2 technique is a universal tool for the high-yield production of heterotrimeric fragments of collagens. Fragments are suitable for mapping functional sites, determining coding sequences of binding sites, elucidating pathogenicity and pathogenic mechanisms of genetic mutations, and production of fragments for protein replacement therapy.
Assuntos
Colágeno Tipo IV , Integrinas , Multimerização Proteica , Animais , Humanos , Sítios de Ligação , Colágeno Tipo IV/química , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Integrinas/química , Integrinas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Mutação , Domínios ProteicosRESUMO
Inhibitors of the ATPase function of bacterial DNA gyrase, located in the GyrB subunit and its related ParE subunit in topoisomerase IV, have demonstrated antibacterial activity. In this study we describe an NMR fragment-based screening effort targeting Staphylococcus aureus GyrB that identified several attractive and novel starting points with good ligand efficiency. Fragment hits were further characterized using NMR binding studies against full-length S. aureus GyrB and Escherichia coli ParE. X-ray co-crystal structures of select fragment hits confirmed binding and suggested a path for medicinal chemistry optimization. The identification, characterization, and elaboration of one of these fragment series to a 0.265 µM inhibitor is described herein.
Assuntos
Antibacterianos/química , Proteínas de Bactérias/antagonistas & inibidores , DNA Girase/química , Inibidores da Topoisomerase II/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/metabolismo , Desenho de Fármacos , Escherichia coli/metabolismo , Ligantes , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/metabolismoRESUMO
Synthesis of bacterial cell wall peptidoglycan requires glycosyltransferase enzymes that transfer the disaccharide-peptide from lipid II onto the growing glycan chain. The polymerization of the glycan chain precedes cross-linking by penicillin-binding proteins and is essential for growth for key bacterial pathogens. As such, bacterial cell wall glycosyltransferases are an attractive target for antibiotic drug discovery. However, significant challenges to the development of inhibitors for these targets include the development of suitable assays and chemical matter that is suited to the nature of the binding site. We developed glycosyltransferase enzymatic activity and binding assays using the natural products moenomycin and vancomycin as model inhibitors. In addition, we designed a library of disaccharide compounds based on the minimum moenomycin fragment with peptidoglycan glycosyltransferase inhibitory activity and based on a more drug-like and synthetically versatile disaccharide building block. A subset of these disaccharide compounds bound and inhibited the glycosyltransferase enzymes, and these compounds could serve as chemical entry points for antibiotic development.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Parede Celular/metabolismo , Peptidoglicano Glicosiltransferase/antagonistas & inibidores , Peptidoglicano/biossíntese , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Desenho de Fármacos , Escherichia coli/enzimologia , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Proteínas de Ligação às Penicilinas/antagonistas & inibidores , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Estrutura Terciária de Proteína , Staphylococcus aureus/efeitos dos fármacos , Vancomicina/química , Vancomicina/metabolismo , Vancomicina/farmacologiaRESUMO
Myxopyronin B (MyxB) binds to the switch region of RNA polymerase (RNAP) and inhibits transcriptional initiation. To evaluate the potential development of MyxB as a novel class of antibiotic, we characterized the antimicrobial activity of MyxB against Staphylococcus aureus. Spontaneous MyxB resistance in S. aureus occurred at a frequency of 8 × 10(-8) , similar to that of rifampin. The MyxB-resistant mutants were found to be altered in single amino acid residues in the RNAP subunits that form the MyxB-binding site. In the presence of human serum albumin, the MyxB minimum inhibitory concentration against S. aureus increased drastically (≥128-fold) and 99.5% of MyxB was protein bound. Because of the high serum protein binding and resistance rate, we conclude that MyxB is not a viable starting point for antibiotic development.
Assuntos
Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Lactonas/farmacologia , Staphylococcus aureus/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Testes de Sensibilidade Microbiana , Mutação , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genéticaRESUMO
Hybrid antimicrobials containing an antibacterial linked to a multidrug resistance (MDR) pump inhibitor make up a promising new class of agents for countering efflux-mediated bacterial drug resistance. This study explores the effects of varying the relative orientation of the antibacterial and efflux pump inhibitor components in three isomeric hybrids (SS14, SS14-M, and SS14-P) which link the antibacterial alkaloid and known substrate for the NorA MDR pump berberine to different positions on INF55 (5-nitro-2-phenylindole), an inhibitor of NorA. The MICs for all three hybrids against wild-type, NorA-knockout, and NorA-overexpressing Staphylococcus aureus cells were found to be similar (9.4 to 40.2 microM), indicating that these compounds are not effectively effluxed by NorA. The three hybrids were also found to have similar curing effects in a Caenorhabditis elegans live infection model. Each hybrid was shown to accumulate in S. aureus cells to a greater extent than either berberine or berberine in the presence of INF55, and the uptake kinetics of SS14 were found to differ from those of SS14-M and SS14-P. The effects on the uptake and efflux of the NorA substrate ethidium bromide into S. aureus cells in the presence or absence of the hybrids were used to confirm MDR inhibition by the hybrids. MDR-inhibitory activity was confirmed for SS14-M and SS14-P but not for SS14. Molecular dynamics simulations revealed that SS14 prefers to adopt a conformation that is not prevalent in either SS14-M or SS14-P, which may explain why some properties of SS14 diverge from those of its two isomers. In summary, subtle repositioning of the pump-blocking INF55 moiety in berberine-INF55 hybrids was found to have a minimal effect on their antibacterial activities but to significantly alter their effects on MDR pumps.
Assuntos
Antibacterianos , Berberina , Enterococcus faecalis/efeitos dos fármacos , Indóis , Staphylococcus aureus/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Berberina/química , Berberina/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla , Etídio , Indóis/química , Indóis/farmacologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Relação Estrutura-AtividadeRESUMO
The nematode Caenorhabditis elegans is a unique whole animal model system for identifying small molecules with in vivo anti-infective properties. C. elegans can be infected with a broad range of human pathogens, including Enterococcus faecalis, an important human nosocomial pathogen. Here, we describe an automated, high-throughput screen of 37,200 compounds and natural product extracts for those that enhance survival of C. elegans infected with E. faecalis. Using a robot to dispense live, infected animals into 384-well plates and automated microscopy and image analysis, we identified 28 compounds and extracts not previously reported to have antimicrobial properties, including six structural classes that cure infected C. elegans animals but do not affect the growth of the pathogen in vitro, thus acting by a mechanism of action distinct from antibiotics currently in clinical use.
Assuntos
Antibacterianos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Modelos Animais de Doenças , Enterococcus faecalis/efeitos dos fármacos , Animais , Antibacterianos/química , Técnicas de Química Combinatória , Avaliação Pré-Clínica de Medicamentos , Enterococcus faecalis/crescimento & desenvolvimento , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Estrutura MolecularRESUMO
The nematode Caenorhabditis elegans shows a high degree of conservation of molecular pathways related to human disease, yet is only 1-mm long and can be considered as a microorganism. Because of the development of a simple but systematic RNA-interference (RNAi) methodology, C. elegans is the only metazoan in which the impact of "knocking-down" nearly every gene in the genome can be analyzed in a whole living animal. Both functional genomic studies and chemical screens can be carried out using C. elegans in vivo screens in a context that preserves intact cell-to-cell communication, neuroendocrine signaling, and every aspect of the animal's metabolism necessary to survive and reproduce in lab conditions. This feature enables studies that are impossible to undertake in cell-culture-based screens. Although genome-wide RNAi screens and limited small-molecule screens have been successfully performed in C. elegans, they are typically extremely labor-intensive. Furthermore, technical limitations have precluded quantitative measurements and time-resolved analyses.In this chapter, we provide detailed protocols to carry out automated high-throughput whole-animal RNAi and chemical screens. We describe methods to perform screens in solid and liquid media, in 96 and 384-well format, respectively. We describe the use of automated handling, sorting, and microscopy of worms. Finally, we give information about worm-adapted image analysis tools to quantify phenotypes. The technology presented here facilitates large-scale C. elegans genetic and chemical screens and it is expected to help shed light on relevant biological areas.
Assuntos
Animais Geneticamente Modificados , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/genética , Interferência de RNA/fisiologia , Animais , Biblioteca Gênica , Taxa de SobrevidaRESUMO
There is an urgent need for the development of new antifungal agents. A facile in vivo model that evaluates libraries of chemical compounds could solve some of the main obstacles in current antifungal discovery. We show that Candida albicans, as well as other Candida species, are ingested by Caenorhabditis elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as filament formation, are also involved in nematode killing. We devised a Candida-mediated C. elegans assay that allows high-throughput in vivo screening of chemical libraries for antifungal activities, while synchronously screening against toxic compounds. The assay is performed in liquid media using standard 96-well plate technology and allows the study of C. albicans in non-planktonic form. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (approximately 1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. Two compounds identified in the screen, caffeic acid phenethyl ester, a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin exhibited antifungal activity in a murine model of candidiasis. The whole-animal C. elegans assay may help to study the molecular basis of C. albicans pathogenesis and identify antifungal compounds that most likely would not be identified by in vitro screens that target fungal growth. Compounds identified in the screen that affect the virulence of Candida in vivo can potentially be used as "probe compounds" and may have antifungal activity against other fungi.
Assuntos
Antifúngicos/farmacologia , Caenorhabditis elegans/microbiologia , Candida/efeitos dos fármacos , Animais , Biofilmes , Ácidos Cafeicos/farmacologia , Enoxacino/farmacologia , Feminino , Fluconazol/farmacologia , Intestinos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/antagonistas & inibidores , Naftoquinonas/farmacologia , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/farmacologiaRESUMO
In bacteria, multidrug-resistance pumps (MDRs) confer resistance to chemically unrelated amphipathic toxins. A major challenge in developing efficacious antibiotics is identifying antimicrobial compounds that are not rapidly pumped out of bacterial cells. The plant antimicrobial berberine, the active component of the medicinal plants echinacea and golden seal, is a cation that is readily extruded by bacterial MDRs, thereby rendering it relatively ineffective as a therapeutic agent. However, inhibition of MDR efflux causes a substantial increase in berberine antimicrobial activity, suggesting that berberine and potentially many other compounds could be more efficacious if an effective MDR pump inhibitor could be identified. Here we show that covalently linking berberine to INF 55 , an inhibitor of Major Facilitator MDRs, results in a highly effective antimicrobial that readily accumulates in bacteria. The hybrid molecule showed good efficacy in a Caenorhabditis elegans model of enterococcal infection, curing worms of the pathogen.
Assuntos
Anti-Infecciosos/farmacologia , Infecções Bacterianas/prevenção & controle , Berberina/química , Farmacorresistência Bacteriana Múltipla , Animais , Infecções Bacterianas/tratamento farmacológico , Fenômenos Fisiológicos Bacterianos , Berberina/farmacologia , Caenorhabditis elegans , Relação Dose-Resposta a Droga , Desenho de Fármacos , Echinacea/metabolismo , Testes de Sensibilidade Microbiana , Modelos Químicos , Extratos Vegetais/metabolismo , Staphylococcus aureus/metabolismoRESUMO
The alarming increase of antibiotic-resistant bacterial pathogens points to the need for novel therapeutic approaches to combat infection. To discover novel antimicrobials, we devised a screen to identify compounds that promoted the survival of the model laboratory nematode Caenorhabditis elegans infected with the human opportunistic pathogen Enterococcus faecalis. E. faecalis colonizes the nematode intestinal tract, forming a persistent lethal infection. Infected nematodes were rescued by antibiotic treatment in a dose-dependent manner, and antibiotic treatment markedly reduced the number of bacteria colonizing the nematode intestine. To facilitate high throughput screening of compound libraries, we adapted a previously developed agar-based C. elegans-E. faecalis infection assay so that it could be carried out in liquid medium in standard 96-well microtiter plates. We used this simple infection system to screen 6,000 synthetic compounds and 1,136 natural product extracts. We identified 16 compounds and 9 extracts that promoted nematode survival. Some of the compounds and extracts inhibited E. faecalis growth in vitro, but, in contrast to traditional antibiotics, the in vivo effective dose of many of these compounds was significantly lower than the minimum inhibitory concentration needed to prevent the growth of E. faecalis in vitro. Moreover, many of the compounds and extracts had little or no affect on in vitro bacterial growth. Our findings indicate that the whole-animal C. elegans screen identifies not only traditional antibiotics, but also compounds that target bacterial virulence or stimulate host defense.
Assuntos
Antibacterianos/farmacologia , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Enterococcus faecalis/efeitos dos fármacos , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Animais , Antibacterianos/análise , Antibacterianos/química , Antibacterianos/uso terapêutico , Caenorhabditis elegans/genética , Caenorhabditis elegans/imunologia , Meios de Cultura , Enterococcus faecalis/fisiologia , Infecções por Bactérias Gram-Positivas/genética , Infecções por Bactérias Gram-Positivas/microbiologia , Infecções por Bactérias Gram-Positivas/patologia , Estrutura Molecular , Mutação/genética , Taxa de SobrevidaRESUMO
Although the opportunistic bacterial pathogen Enterococcus faecium is a leading source of nosocomial infections, it appears to lack many of the overt virulence factors produced by other bacterial pathogens, and the underlying mechanism of pathogenesis is not clear. Using E. faecium-mediated killing of the nematode worm Caenorhabditis elegans as an indicator of toxicity, we determined that E. faecium produces hydrogen peroxide at levels that cause cellular damage. We identified E. faecium transposon insertion mutants with altered C. elegans killing activity, and these mutants were altered in hydrogen peroxide production. Mutation of an NADH oxidase-encoding gene eliminated nearly all NADH oxidase activity and reduced hydrogen peroxide production. Mutation of an NADH peroxidase-encoding gene resulted in the enhanced accumulation of hydrogen peroxide. E. faecium is able to produce hydrogen peroxide by using glycerol-3-phosphate oxidase, and addition of glycerol to the culture medium enhanced the killing of C. elegans. Conversely, addition of glucose, which leads to the down-regulation of glycerol metabolism, prevented both C. elegans killing and hydrogen peroxide production. Lastly, detoxification of hydrogen peroxide either by exogenously added catalase or by a C. elegans transgenic strain overproducing catalase prevented E. faecium-mediated killing. These results suggest that hydrogen peroxide produced by E. faecium has cytotoxic effects and highlight the utility of C. elegans pathogenicity models for identifying bacterial virulence factors.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Enterococcus faecium/patogenicidade , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Elementos de DNA Transponíveis , Enterococcus faecium/genética , Enterococcus faecium/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Peroxidases/genética , VirulênciaRESUMO
In eukaryotes, mRNAs are transcribed in the nucleus and exported to the cytoplasm for translation to occur. Messenger RNAs complexed with proteins referred to as ribonucleoparticles are recognized for nuclear export in part by association with Mex67, a key Saccharomyces cerevisiae mRNA export factor and homolog of human TAP/NXF1. Mex67, along with its cofactor Mtr2, is thought to promote ribonucleoparticle translocation by interacting directly with components of the nuclear pore complex (NPC). Herein, we show that the nuclear pore-associated protein Sac3 functions in mRNA export. Using a mutant allele of MTR2 as a starting point, we have identified a mutation in SAC3 in a screen for synthetic lethal interactors. Loss of function of SAC3 causes a strong nuclear accumulation of mRNA and synthetic lethality with a number of mRNA export mutants. Furthermore, Sac3 can be coimmunoprecipitated with Mex67, Mtr2, and other factors involved in mRNA export. Immunoelectron microscopy analysis shows that Sac3 localizes exclusively to cytoplasmic fibrils of the NPC. Finally, Mex67 accumulates at the nuclear rim when SAC3 is mutated, suggesting that Sac3 functions in Mex67 translocation through the NPC.
Assuntos
Citoesqueleto/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Biológico/fisiologia , Humanos , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Porinas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants, we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Eucarióticas/metabolismo , RNA Ribossômico/metabolismo , Receptores Citoplasmáticos e Nucleares , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae , Bioensaio/métodos , Células Cultivadas , Proteínas Ativadoras de GTPase , Regulação Fúngica da Expressão Gênica/genética , Carioferinas/genética , Carioferinas/metabolismo , Mutação/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , RNA Ribossômico/genética , Ribossomos/genética , Saccharomyces cerevisiae , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Proteína Exportina 1RESUMO
Saccharomyces cerevisiae CGR1 encodes a conserved fungal protein that localizes to the nucleolus. To determine if this localization reflects a role for Cgr1p in ribosome biogenesis two yeast cgr1 mutants were examined for defects in ribosome synthesis: a conditional depletion strain in which CGR1 is under the control of a tetracycline-repressible promoter and a mutant strain in which a C-terminal truncated Cgr1p is expressed. Both strains had impaired growth rates and were hypersensitive to the aminoglycosides paromomycin and hygromycin. Polysome analyses of the mutants revealed increased levels of free 40S subunits relative to 60S subunits, a decrease in 80S monosomes and accumulation of half-mer polysomes. Pulse-chase labelling demonstrated that pre-rRNA processing was defective in the mutants, resulting in accumulation of the 35S, 27S and 7S pre-rRNAs and delayed production of the mature 25S and 5 small middle dot8S rRNAs. The synthesis of the 18S and 5S rRNAs was unaffected. Loss of Cgr1 function also caused a partial delocalization of the 5'-ITS1 RNA and the nucleolar protein Nop1p into the nucleoplasm, suggesting that Cgr1p contributes to compartmentalization of nucleolar constituents. Together these findings establish a role for Cgr1p in ribosome biogenesis.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Fúngico/genética , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Bases , Nucléolo Celular/genética , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico/genética , Proteínas Recombinantes/metabolismoRESUMO
The cellular response to environmental changes includes widespread modifications in gene expression. Here we report the identification and characterization of Rsc9, a member of the RSC chromatin-remodeling complex in yeast. The genome-wide localization of Rsc9 indicated a relationship between genes targeted by Rsc9 and genes regulated by stress; treatment with hydrogen peroxide or rapamycin, which inhibits TOR signaling, resulted in genome-wide changes in Rsc9 occupancy. We further show that Rsc9 is involved in both repression and activation of mRNAs regulated by TOR as well as the synthesis of rRNA. Our results illustrate the response of a chromatin-remodeling factor to signaling cascades and suggest that changes in the activity of chromatin-remodeling factors are reflected in changes in their localization in the genome.