RESUMEN
The TRIM family of genes is largely studied because of their roles in development, differentiation and host cell antiviral defenses; however, roles in cancer biology are emerging. Loss of heterozygosity of the TRIM3 locus in â¼20% of human glioblastomas raised the possibility that this NHL-domain containing member of the TRIM gene family might be a mammalian tumor suppressor. Consistent with this, reducing TRIM3 expression increased the incidence of and accelerated the development of platelet-derived growth factor -induced glioma in mice. Furthermore, TRIM3 can bind to the cdk inhibitor p21(WAF1/CIP1). Thus, we conclude that TRIM3 is a tumor suppressor mapping to chromosome 11p15.5 and that it might block tumor growth by sequestering p21 and preventing it from facilitating the accumulation of cyclin D1-cdk4.
Asunto(s)
Proteínas Portadoras/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Glioblastoma/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Línea Celular Transformada , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/patología , Humanos , Immunoblotting , Pérdida de Heterocigocidad , Ratones , Ratones Noqueados , Mutación , Unión Proteica , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Eighteen histone deacetylases (HDACs) are present in humans, categorized into two groups: zinc-dependent enzymes (HDAC1-11) and NAD(+)-dependent enzymes (sirtuins 1-7). Among zinc-dependent HDACs, HDAC6 is unique. It has a cytoplasmic localization, two catalytic sites, a ubiquitin-binding site, and it selectively deacetylases alpha-tubulin and Hsp90. Here, we report the discovery that the redox regulatory proteins, peroxiredoxin (Prx) I and Prx II are specific targets of HDAC6. Prx are antioxidants enzymes whose main function is H(2)O(2) reduction. Prx are elevated in many cancers and neurodegenerative diseases. The acetylated form of Prx accumulates in the absence of an active HDAC6. Acetylation of Prx increases its reducing activity, its resistance to superoxidation, and its resistance to transition to high-molecular-mass complexes. Thus, HDAC6 and Prx are targets for modulating intracellular redox status in therapeutic strategies for disorders as disparate as cancers and neurodegenerative diseases.
Asunto(s)
Histona Desacetilasas/metabolismo , Peroxirredoxinas/metabolismo , Acetilación , Línea Celular Tumoral , Histona Desacetilasa 6 , Histona Desacetilasas/análisis , Humanos , Oxidación-Reducción , Estrés Oxidativo , Peróxidos/metabolismoRESUMEN
Mycobacterium tuberculosis (Mtb) mounts a stubborn defense against oxidative and nitrosative components of the immune response. Dihydrolipoamide dehydrogenase (Lpd) and dihydrolipoamide succinyltransferase (SucB) are components of alpha-ketoacid dehydrogenase complexes that are central to intermediary metabolism. We find that Lpd and SucB support Mtb's antioxidant defense. The peroxiredoxin alkyl hydroperoxide reductase (AhpC) is linked to Lpd and SucB by an adaptor protein, AhpD. The 2.0 angstrom AhpD crystal structure reveals a thioredoxin-like active site that is responsive to lipoamide. We propose that Lpd, SucB (the only lipoyl protein detected in Mtb), AhpD, and AhpC together constitute a nicotinamide adenine dinucleotide (reduced)-dependent peroxidase and peroxynitrite reductase. AhpD thus represents a class of thioredoxin-like molecules that enables an antioxidant defense.
Asunto(s)
Aciltransferasas/metabolismo , Dihidrolipoamida Deshidrogenasa/metabolismo , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/metabolismo , Peroxidasas/química , Peroxidasas/metabolismo , Ácido Tióctico/análogos & derivados , Secuencia de Aminoácidos , Antioxidantes , Sitios de Unión , Catálisis , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Enlace de Hidrógeno , Peróxido de Hidrógeno/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , NAD/metabolismo , Oxidación-Reducción , Peroxirredoxinas , Ácido Peroxinitroso/metabolismo , Conformación Proteica , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Ácido Tióctico/metabolismo , Tiorredoxinas/química , Tiorredoxinas/metabolismoRESUMEN
We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Leucocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Factor 6 de Ribosilación del ADP , Animales , Células COS , Proteínas Portadoras/genética , Clonación Molecular , Citosol/metabolismo , Proteínas Activadoras de GTPasa/genética , Leucocitos/ultraestructura , Espectrometría de Masas , Datos de Secuencia Molecular , Unión Proteica , Proteínas/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , PorcinosRESUMEN
Sin3 is an evolutionarily conserved corepressor that exists in different complexes with the histone deacetylases HDAC1 and HDAC2. Sin3-HDAC complexes are believed to deacetylate nucleosomes in the vicinity of Sin3-regulated promoters, resulting in a repressed chromatin structure. We have previously found that a human Sin3-HDAC complex includes HDAC1 and HDAC2, the histone-binding proteins RbAp46 and RbAp48, and two novel polypeptides SAP30 and SAP18. SAP30 is a specific component of Sin3 complexes since it is absent in other HDAC1/2-containing complexes such as NuRD. SAP30 mediates interactions with different polypeptides providing specificity to Sin3 complexes. We have identified p33ING1b, a negative growth regulator involved in the p53 pathway, as a SAP30-associated protein. Two distinct Sin3-p33ING1b-containing complexes were isolated, one of which associates with the subunits of the Brg1-based Swi/Snf chromatin remodeling complex. The N terminus of p33ING1b, which is divergent among a family of ING1 polypeptides, associates with the Sin3 complex through direct interaction with SAP30. The N-terminal domain of p33 is present in several uncharacterized human proteins. We show that overexpression of p33ING1b suppresses cell growth in a manner dependent on the intact Sin3-HDAC-interacting domain.
Asunto(s)
División Celular/fisiología , Proteínas de Drosophila , Inhibidores de Crecimiento/metabolismo , Histona Desacetilasas/metabolismo , Proteínas/metabolismo , Proteínas de Unión al ARN , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas de Ciclo Celular , Línea Celular , ADN Helicasas , ADN Complementario/genética , Proteínas de Unión al ADN , Genes Supresores de Tumor , Inhibidores de Crecimiento/genética , Células HeLa , Histona Desacetilasa 2 , Histona Desacetilasas/química , Humanos , Proteína Inhibidora del Crecimiento 1 , Péptidos y Proteínas de Señalización Intracelular , Sustancias Macromoleculares , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Homología de Secuencia de Aminoácido , Complejo Correpresor Histona Desacetilasa y Sin3 , Factores de Transcripción/química , Proteínas Supresoras de TumorRESUMEN
FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line
Asunto(s)
Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas/metabolismo , Androstadienos/farmacología , Animales , Brefeldino A/farmacología , Células COS , Proteínas Portadoras/química , Proteínas Portadoras/genética , Endosomas/química , Endosomas/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Aparato de Golgi/efectos de los fármacos , Proteínas de la Matriz de Golgi , Proteínas Fluorescentes Verdes , Humanos , Indicadores y Reactivos/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Lípidos de la Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas , Proteínas/química , Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Resonancia por Plasmón de Superficie , Wortmanina , Dedos de ZincRESUMEN
The trithorax genes encode an evolutionarily conserved family of proteins that function to maintain specific patterns of gene expression throughout cellular development. Members of this protein family contain a highly conserved 130- to 140-amino acid motif termed the SET domain. We report the purification and molecular identification of the subunits of a protein complex in the yeast Saccharomyces cerevisiae that includes the trithorax-related protein Set1. This protein complex, which we have named COMPASS (Complex Proteins Associated with Set1), consists of seven polypeptides ranging from 130 to 25 kDa. The same seven proteins were identified in COMPASS purified either by conventional biochemical chromatography or tandem-affinity tagging of the individual subunits of the complex. Null mutants missing any one of the six nonessential subunits of COMPASS grow more slowly than wild-type cells under normal conditions and demonstrate growth sensitivity to hydroxyurea. Furthermore, gene expression profiles of strains missing either of two nonessential subunits of COMPASS are altered in similar ways, suggesting these proteins have similar roles in gene expression in vivo. Molecular characterization of trithorax complexes will facilitate defining the role of this class of proteins in the regulation of gene expression and how their misregulation results in the development of human cancer.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Cromatografía Liquida , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica , Silenciador del Gen , N-Metiltransferasa de Histona-Lisina , Hidroxiurea/farmacología , Mutación , Fenotipo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/aislamiento & purificación , Telómero , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificaciónRESUMEN
In Saccharomyces cerevisiae, heme directly mediates the effects of oxygen on transcription through the heme activator protein Hap1. In the absence of heme, Hap1 is bound by at least four cellular proteins, including Hsp90 and Ydj1, forming a higher-order complex, termed HMC, and its activity is repressed. Here we purified the HMC and showed by mass spectrometry that two previously unidentified major components of the HMC are the Ssa-type Hsp70 molecular chaperone and Sro9 proteins. In vivo functional analysis, combined with biochemical analysis, strongly suggests that Ssa proteins are critical for Hap1 repression in the absence of heme. Ssa may repress the activities of both Hap1 DNA-binding and activation domains. The Ssa cochaperones Ydj1 and Sro9 appear to assist Ssa in Hap1 repression, and only Ydj1 residues 1 to 172 containing the J domain are required for Hap1 repression. Our results suggest that Ssa-Ydj1 and Sro9 act together to mediate Hap1 repression in the absence of heme and that molecular chaperones promote heme regulation of Hap1 by a mechanism distinct from the mechanism of steroid signaling.
Asunto(s)
Proteínas de Unión al ADN , Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Hemo/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Transactivadores/metabolismo , Western Blotting , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Sustancias Macromoleculares , Espectrometría de Masas , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/metabolismo , Mutación , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae , Eliminación de Secuencia , Transactivadores/genética , Factores de TranscripciónRESUMEN
MEF, a recently identified member of the E74 family of ETS-related transcription factors, is a strong transcriptional activator of cytokine gene expression. Using a green fluorescent protein gene reporter plasmid regulated by an MEF-responsive promoter, we determined that the transcriptional activity of MEF is largely restricted to the G1 phase of the cell cycle. MEF-dependent transcription was suppressed by the expression of cyclin A but not by cyclin D or cyclin E. This effect was due to the kinase activity generated by cyclin A expression, as co-expression of the cyclin-dependent kinase inhibitors p21 or p27, or a dominant negative form of CDK2 (DNK2), abrogated the reduction of MEF transcriptional activity by cyclin A. Cyclin A-CDK2 phosphorylated MEF protein in vitro more efficiently than cyclin D-CDK4 or cyclin E-CDK2, and phosphorylation of MEF by cyclin A-CDK2 reduced its ability to bind DNA. We determined one site of phosphorylation by cyclin A-CDK2 at the C terminus of MEF, using mass-spectrometry; mutation of three serine or threonine residues in this region significantly reduced phosphorylation of MEF by cyclin A and reduced cyclin A-mediated suppression of its transactivating activity. These amino acid substitutions also reduced the restriction of MEF activity to G1. Phosphorylation of MEF by the cyclin A-CDK2 complex controls its transcriptional activity during the cell cycle, establishing a novel link between the ETS family of proteins and the cell cycle machinery.
Asunto(s)
Ciclina A/metabolismo , Proteínas de Unión al ADN/metabolismo , Fase G1/fisiología , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Cartilla de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Datos de Secuencia Molecular , Fosforilación , Factores de Transcripción/química , Factores de Transcripción/fisiología , Transcripción GenéticaRESUMEN
A goal of modern biology is to identify the physical interactions that define 'functional modules' of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate 'on' or 'off' state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes, but these complexes have not been described owing to inherent difficulties in purification. We previously fractionated a major PcG complex, PRC1, to 20-50% homogeneity from Drosophila embryos. Here, we identify 30 proteins in these preparations, then further fractionate the preparation and use western analyses to validate unanticipated connections. We show that the known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery.
Asunto(s)
Proteínas de Unión al ADN/análisis , Proteínas de Drosophila , Proteínas de Insectos/química , Factores de Transcripción TFII/análisis , Animales , Western Blotting , Cromatografía en Gel , ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Drosophila , Regulación de la Expresión Génica , Genes de Insecto , Proteínas de Insectos/fisiología , Espectrometría de Masas , Complejo Represivo Polycomb 1 , Pruebas de Precipitina , Unión Proteica , Factor de Transcripción TFIID , Factores de Transcripción TFII/fisiología , Transcripción GenéticaRESUMEN
Inducible nitric oxide synthase (iNOS) plays an important role in host defense. Macrophages expressing iNOS release the reactive nitrogen intermediates (RNI) nitrite and S-nitrosoglutathione (GSNO), which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages. We sought to characterize the active intrabacterial forms of these RNI and their molecular targets. Peptide methionine sulfoxide reductase (MsrA; EC ) catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met). E. coli lacking MsrA were hypersensitive to killing not only by hydrogen peroxide, but also by nitrite and GSNO. The wild-type phenotype was restored by transformation with plasmids encoding msrA from E. coli or M. tuberculosis, but not by an enzymatically inactive mutant msrA, indicating that Met oxidation was involved in the death of these cells. It seemed paradoxical that nitrite and GSNO kill bacteria by oxidizing Met residues when these RNI cannot themselves oxidize Met. However, under anaerobic conditions, neither nitrite nor GSNO was bactericidal. Nitrite and GSNO can both give rise to NO, which may react with superoxide produced by bacteria during aerobic metabolism, forming peroxynitrite, a known oxidant of Met to Met-O. Thus, the findings are consistent with the hypotheses that nitrite and GSNO kill E. coli by intracellular conversion to peroxynitrite, that intracellular Met residues in proteins constitute a critical target for peroxynitrite, and that MsrA can be essential for the repair of peroxynitrite-mediated intracellular damage.
Asunto(s)
Proteínas Bacterianas/fisiología , Escherichia coli/enzimología , Mycobacterium tuberculosis/enzimología , Oxidorreductasas/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/metabolismo , Prueba de Complementación Genética , Glutatión/análogos & derivados , Glutatión/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Metionina/metabolismo , Metionina Sulfóxido Reductasas , Mycobacterium tuberculosis/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Compuestos Nitrosos/metabolismo , Estrés Oxidativo , Oxidorreductasas/genética , Fenotipo , Proteínas Recombinantes de Fusión/metabolismo , S-NitrosoglutatiónRESUMEN
Cyclin-dependent kinase (CDK)7-cyclin H, the CDK-activating kinase (CAK) and TFIIH-associated kinase in metazoans can be activated in vitro through T-loop phosphorylation or binding to the RING finger protein MAT1. Although the two mechanisms can operate independently, we show that in a physiological setting, MAT1 binding and T-loop phosphorylation cooperate to stabilize the CAK complex of Drosophila. CDK7 forms a stable complex with cyclin H and MAT1 in vivo only when phosphorylated on either one of two residues (Ser164 or Thr170) in its T-loop. Mutation of both phosphorylation sites causes temperature-dependent dissociation of CDK7 complexes and lethality. Furthermore, phosphorylation of Thr170 greatly stimulates the activity of the CDK7- cyclin H-MAT1 complex towards the C-terminal domain of RNA polymerase II without significantly affecting activity towards CDK2. Remarkably, the substrate-specific increase in activity caused by T-loop phosphorylation is due entirely to accelerated enzyme turnover. Thus phosphorylation on Thr170 could provide a mechanism to augment CTD phosphorylation by TFIIH-associated CDK7, and thereby regulate transcription.
Asunto(s)
Quinasas Ciclina-Dependientes , Ciclinas/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Biopolímeros , Ciclina H , Ciclinas/antagonistas & inhibidores , Drosophila , Proteínas de Drosophila , Cinética , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Especificidad por Sustrato , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX domain.
Asunto(s)
Neutrófilos/enzimología , Oxidorreductasas/sangre , Oxidorreductasas/efectos de los fármacos , Fosfatos de Fosfatidilinositol/farmacología , Fosfoproteínas/metabolismo , Animales , Sitios de Unión , Grupo Citocromo b/efectos de los fármacos , Grupo Citocromo b/metabolismo , Membranas Artificiales , Oxidación-Reducción , Fosfoproteínas/química , Estructura Terciaria de Proteína , Superóxidos/metabolismo , PorcinosRESUMEN
Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.
Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Acetiltransferasas/genética , Acetiltransferasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Clonación Molecular , Secuencia Conservada , Drosophila melanogaster/genética , Histona Acetiltransferasas , Humanos , Sustancias Macromoleculares , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fenotipo , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Ácidos Grasos Insaturados/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana , Transducción de Señal , Familia-src Quinasas/metabolismo , Acilación , Animales , Células COS , Proteínas Portadoras/metabolismo , Humanos , Células Jurkat , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-fyn , ConejosRESUMEN
A three-subunit Hap complex that interacts with the RNA polymerase II Elongator was isolated from yeast. Deletions of genes for two Hap subunits, HAP1 and HAP3, confer pGKL killer-insensitive and weak Elongator phenotypes. Preferential interaction of the Hap complex with free rather than RNA polymerase II-associated Elongator suggests a role in the regulation of Elongator activity.
Asunto(s)
ADN-(Sitio Apurínico o Apirimidínico) Liasa , ARN Polimerasa II/química , ARN Polimerasa II/aislamiento & purificación , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos , Factor de Unión a CCAAT/genética , Liasas de Carbono-Oxígeno/genética , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Fenotipo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Saccharomyces cerevisiae/enzimología , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
Acetylation of core histone tails plays a fundamental role in transcription regulation. In addition to acetylation, other posttranslational modifications, such as phosphorylation and methylation, occur in core histone tails. Here, we report the purification, molecular identification, and functional characterization of a histone H4-specific methyltransferase PRMT1, a protein arginine methyltransferase. PRMT1 specifically methylates arginine 3 (Arg 3) of H4 in vitro and in vivo. Methylation of Arg 3 by PRMT1 facilitates subsequent acetylation of H4 tails by p300. However, acetylation of H4 inhibits its methylation by PRMT1. Most important, a mutation in the S-adenosyl-l-methionine-binding site of PRMT1 substantially crippled its nuclear receptor coactivator activity. Our finding reveals Arg 3 of H4 as a novel methylation site by PRMT1 and indicates that Arg 3 methylation plays an important role in transcriptional regulation.
Asunto(s)
Arginina/metabolismo , Histonas/metabolismo , Metiltransferasas/metabolismo , Receptores Androgénicos/metabolismo , Activación Transcripcional , Acetilación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Núcleo Celular/metabolismo , Células HeLa , Histonas/química , Humanos , Ácidos Hidroxámicos/farmacología , Péptidos y Proteínas de Señalización Intracelular , Lisina/metabolismo , Metilación , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/aislamiento & purificación , Datos de Secuencia Molecular , Mutación , Oocitos , Proteína-Arginina N-Metiltransferasas , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/metabolismo , XenopusRESUMEN
Chromatin remodeling complexes perform central roles in transcriptional regulation. Here, we identify Rsc3 and Rsc30 as novel components of the essential yeast remodeler RSC complex. Rsc3 and Rsc30 function requires their zinc cluster domain, a known site-specific DNA binding motif. RSC3 is essential, and rsc3 Ts- mutants display a G2/M cell cycle arrest involving the spindle assembly checkpoint pathway, whereas rsc30Delta mutants are viable and osmosensitive. Rsc3 and Rsc30 interact functionally and also physically as a stable Rsc3/Rsc30 heteromeric complex. However, DNA microarray analysis with rsc3 or rsc30 mutants reveals different effects on the expression levels of ribosomal protein genes and cell wall genes. We propose that Rsc3 and Rsc30 interact physically but have different roles in targeting or regulating RSC.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae , Factores de Transcripción/genética , Zinc/metabolismo , Secuencia de Bases , Ciclo Celular/genética , Pared Celular/fisiología , Cromatina/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Fase G2/genética , Genes Letales/fisiología , Genes cdc/fisiología , Leucina Zippers/fisiología , Mitosis/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Huso Acromático/genética , Huso Acromático/metabolismo , Temperatura , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Levaduras , Zinc/químicaRESUMEN
We describe a microflow-based instrument, consisting of multiple rotary valves, capillary tubing, and miniaturized reaction vessels, for the purpose of performing automated chemical and biochemical reations on a very small scale (i.e. submicroliter volumes). The novelty is that close to 100% of the reaction end products are available in a minimal volume (< or = 5 microL) inside a pressurized microvial for subsequent analysis. This makes the system compatible with capillary HPLC and, in principle, with continuous-flow nanoelectrospray mass spectrometry. Total control of flow path combinations and directions, temperatures, and gas pressures enables precise execution of complex biochemical laboratory procedures. Instrument performance was convincingly demonstrated by partially sequencing 100 fmol of an intact protein using classical Edman chemistry in combination with capillary-bore liquid chromatography. To our knowledge, this is the smallest amount of protein ever reported to be successfully analyzed in this way. Additional applications that merge enzymatic and chemical procedures with high-sensitivity analytical devices can be envisioned in the future.