RESUMEN
Hereditary spastic paraplegia (HSP) is a neurodegenerative disorder selectively affecting axons of spinal cord motoneurons. Classical mutations in the most frequent HSP gene SPAST (spastin protein) act through haploinsufficiency by abolishing the activity of a C-terminal ATPase domain or by interfering with expression from the affected allele. N-terminal missense variants have been suggested to represent rare polymorphisms, to cause unusually mild phenotypes, and to aggravate the effect of a classical mutation. We confirm these associations for p.S44L but do not detect two other variants (p.E43Q; p.P45Q) in HSP patients and controls. We show that neither of several disease mechanisms associated with classical SPAST mutations applies to the N-terminal variants. Instead, all three alterations enhance the stability of one of two alternative spastin isoforms. Their phenotypic effect may thus not be mediated by haploinsufficiency but by increasing isoform competition for interacting proteins, substrates or oligomerization partners.
Asunto(s)
Adenosina Trifosfatasas/genética , Predisposición Genética a la Enfermedad/genética , Mutación Missense/genética , Paraplejía Espástica Hereditaria/genética , Paraplejía Espástica Hereditaria/metabolismo , Adolescente , Adulto , Edad de Inicio , Empalme Alternativo , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Frecuencia de los Genes , Pruebas Genéticas , Variación Genética , Genotipo , Haplotipos , Humanos , Masculino , Linaje , Fenotipo , Polimorfismo Genético , Isoformas de Proteínas/genética , Estructura Terciaria de Proteína/genética , Paraplejía Espástica Hereditaria/fisiopatología , EspastinaRESUMEN
Angiogenesis is a prerequisite for solid tumor growth and metastasis. Elucidation of the signaling pathways that control tumor angiogenesis constitutes the basis for a rational antiangiogenic tumor therapy. Here we show that the production of vascular endothelial growth factor (VEGF) in HeLa and HL-60 cells is directed by the constitutive photomorphogenesis 9 signalosome (CSN). The CSN is a kinase complex that cooperates with the ubiquitin/26S proteasome system in regulating the stability of proteins involved in signal transduction. VEGF expression is controlled by the transcription factors activator protein (AP)-1, AP-2, SP-1, and hypoxia-inducible factor 1. Inhibition of CSN kinase activity by 50 microM curcumin for 2 h decreases the cellular c-Jun concentration, resulting in a reduction of the VEGF production by approximately 75%. The removal of the inhibitor from the cells led to a time-dependent recovery of endogenous c-Jun that is paralleled by increasing VEGF production. Elevated cellular CSN activity induced by CSN subunit 2 overexpression causes increased VEGF production in HeLa cells. A competitor of CSN-dependent c-Jun phosphorylation, the NH(2)-terminal c-Jun fragment Deltac-Jun(1-226), inhibits VEGF production in HeLa cells. The transcription factors AP-2 and SP-1 act independently of the CSN. They contribute less than a quarter to basal VEGF production. Under our experimental conditions, hypoxia-inducible factor 1alpha protein was not detected. Overexpression of the tumor suppressor p53 reduces VEGF production in HeLa cells. p53 competes with c-Jun for CSN-specific phosphorylation with the consequence of c-Jun destabilization. We conclude that CSN-directed c-Jun signaling mediates high VEGF production in HeLa and HL-60 cells. The data provide an explanation for the known antiangiogenic and antitumorigenic activities of curcumin. Because the CSN regulates the major part of VEGF production in the tested tumor cells, it constitutes a potentially important target for tumor therapy.
Asunto(s)
Factores de Crecimiento Endotelial/biosíntesis , Linfocinas/biosíntesis , Proteínas/fisiología , Complejo del Señalosoma COP9 , Proteínas de Unión al ADN/metabolismo , Células HL-60/enzimología , Células HL-60/metabolismo , Células HeLa , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Complejos Multiproteicos , Péptido Hidrolasas , Transducción de Señal/fisiología , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción AP-2 , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/farmacología , Proteína p53 Supresora de Tumor/fisiología , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The COP9 signalosome is a multiprotein complex somewhat similar to the lid component of the 26S proteasome. Recent studies suggest that it regulates the stability of proteins by interfering with the ubiquitin-proteasome pathway via deneddylation and phosphorylation.
Asunto(s)
Proteínas/metabolismo , Transducción de Señal/fisiología , Complejo del Señalosoma COP9 , Sustancias Macromoleculares , Modelos Biológicos , Complejos Multiproteicos , Péptido Hidrolasas , Fosforilación , Proteínas de Plantas/metabolismo , Subunidades de Proteína , Proteínas/genética , Ubiquitinas/metabolismoRESUMEN
In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.
Asunto(s)
Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Complejo del Señalosoma COP9 , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HL-60 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Complejos Multiproteicos , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Treonina/genética , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/genética , Valina/genéticaRESUMEN
Interferon consensus sequence-binding protein (ICSBP) is a member of the interferon regulatory factors (IRF) that has a pivotal role in mediating resistance to pathogenic infections in mice and in promoting the differentiation of myeloid cells. ICSBP exerts some of its transcriptional activities via association with other factors that enable its binding to a variety of promoters containing DNA composite elements. These interactions are mediated through a specific COOH-terminal domain termed IAD (IRF association domain). To gain a broader insight of the capacity of ICSBP to interact with other factors, yeast two-hybrid screens were performed using ICSBP-IAD as a bait against a B-cell cDNA library. Trip15 was identified as a specific interacting factor with ICSBP in yeast cells, which was also confirmed by in vitro glutathione S-transferase pull-down assays and by coimmunoprecipitation studies in COS7 cells. Trip15 was recently identified as a component of the COP9/signalosome (CSN) complex composed of eight evolutionary conserved subunits and thus termed CSN2. This complex has a role in cell-signaling processes, which is manifested by its associated novel kinase activity and by the involvement of its subunits in regulating multiple cell-signaling pathways and cell-cycle progression. We show that in vitro association of ICSBP with the CSN leads to phosphorylation of ICSBP at a unique serine residue within its IAD. The phosphorylated residue is essential for efficient association with IRF-1 and thus for the repressor activity of ICSBP exerted on IRF-1. This suggests that the CSN has a role in integrating incoming signals that affect the transcriptional activity of ICSBP.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas Nucleares , Proteínas/química , Proteínas/metabolismo , Receptores de Hormona Tiroidea , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Factores de Transcripción , Células 3T3 , Alanina/química , Animales , Linfocitos B/metabolismo , Northern Blotting , Complejo del Señalosoma COP9 , Células COS , ADN/metabolismo , ADN Complementario/metabolismo , Relación Dosis-Respuesta a Droga , Biblioteca de Genes , Glutatión Transferasa/metabolismo , Células HL-60 , Células HeLa , Humanos , Factores Reguladores del Interferón , Ratones , Modelos Biológicos , Complejos Multiproteicos , Mutagénesis Sitio-Dirigida , Péptido Hidrolasas , Fosforilación , Plásmidos/metabolismo , Pruebas de Precipitina , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Serina/química , Transducción de Señal , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
The COP9 signalosome is involved in signal transduction, whereas the 26 S proteasome lid is a regulatory subcomplex of the 26 S proteasome responsible for degradation of ubiquitinated proteins. COP9 signalosome and lid possess significant sequence homologies among their eight core subunits and are likely derived from a common ancestor. Surprisingly, from our two-dimensional electron microscopy data, a common architectural plan for the two complexes could not be deduced. None-the-less, the two particles have structural features in common. Both COP9 signalosome and lid lack any symmetry in subunit arrangement and exhibit a central groove, possibly qualified for scaffolding functions.Filter-binding assays with recombinant COP9 signalosome components revealed a multitude of subunit-subunit interactions, supporting the asymmetrical appearance of the complex in electron microscopy. On the basis of two-dimensional images and subunit interaction studies, a first architectural model of COP9 signalosome was created. The fact that four distinct classes of particle views were identified and that only 50 % of the selected particles could be classified indicates a high degree of heterogeneity in electron microscopic images. Different orientations with respect to the viewing axis and conformational variety, presumably due to different grades of phosphorylation, are possible reasons for the heterogeneous appearance of the complex. Our biochemical data show that recombinant COP9 signalosome subunits 2 and 7 are phosphorylated by the associated kinase activity. The modification of COP9 signalosome subunit 2 might be essential for c-Jun phosphorylation. Dephosphorylation does not inactivate the associated kinase activity. Although substrate phosphorylation by COP9 signalosome is significantly decreased by lambda protein phosphatase treatment, "autophosphorylation" is increased.
Asunto(s)
Péptido Hidrolasas/metabolismo , Péptido Hidrolasas/ultraestructura , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismo , Proteínas/ultraestructura , Complejo del Señalosoma COP9 , Electroforesis en Gel de Poliacrilamida , Eritrocitos/química , Eritrocitos/enzimología , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica , Complejos Multiproteicos , Péptido Hidrolasas/química , Fosforilación , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas/química , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Transducción de SeñalRESUMEN
Substrates are targeted for proteolysis by the ubiquitin pathway by the addition of a polyubiquitin chain before being degraded by the 26 S proteasome. Previously, a subunit of the proteasome, S5a, was identified that was able to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component. Deletion of the corresponding Saccharomyces cerevisiae gene, MCB1/RPN10, rendered cells viable indicating that other proteasomal polyubiquitin receptors must exist. In this study, we describe pus1(+), the fission yeast homologue of RPN10. This gene is also not required for cell viability; however, the Deltapus1 mutant is synthetically lethal with mutations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1). Overexpression of pus1(+) is able to rescue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly reduced viability when used to rescue the mts3-1Deltapus1 double mutant. The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 and Mts3 proteins were found to bind to one another in vitro, which taken together with the genetic data suggests that they are also closely associated in vivo.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Sitios de Unión , Proteínas Portadoras/química , Drosophila melanogaster/genética , Endopeptidasas/metabolismo , Eliminación de Gen , Humanos , Hidroliasas/genética , Hidroliasas/metabolismo , Datos de Secuencia Molecular , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteínas de Unión al ARN , Saccharomyces cerevisiae/genética , Schizosaccharomyces/crecimiento & desarrollo , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The 26S proteasome is the major non-lysosomal protease in eukaryotic cells. This multimeric enzyme is the integral component of the ubiquitin-mediated substrate degradation pathway. It consists of two subcomplexes, the 20S proteasome, which forms the proteolytic core, and the 19S regulator (or PA700), which confers ATP dependency and ubiquitinated substrate specificity on the enzyme. Recent biochemical and genetic studies have revealed many of the interactions between the 17 regulatory subunits, yielding an approximation of the 19S complex topology. Inspection of interactions of regulatory subunits with non-subunit proteins reveals patterns that suggest these interactions play a role in 26S proteasome regulation and localization.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Humanos , Proteínas/metabolismo , Proteínas de Unión al ARN , Especificidad de la EspecieRESUMEN
The basic region-leucine zipper transcription factor c-Jun regulates gene expression and cell function. It participates in the formation of homo- or heterodimeric complexes that specifically bind to DNA sequences called activating protein 1 (AP-1) sites. The stability and activity of c-Jun is regulated by phosphorylation within the N-terminal activation domain. Mitogen- and stress-activated c-Jun N-terminal kinases (JNKs) were previously the only described enzymes phosphorylating c-Jun at the N terminus in vivo. In this report we demonstrate a JNK-independent activation of c-Jun in vivo directed by the constitutive photomorphogenesis (COP9) signalosome. The overexpression of signalosome subunit 2 (Sgn2), a subunit of the COP9 signalosome, leads to de novo assembly of the complex with a partial incorporation of the overexpressed subunit. The de novo formation of COP9 signalosome parallels an increase of c-Jun protein resulting in elevated AP-1 transcriptional activity. The c-Jun activation caused by Sgn2 overexpression is independent of JNK and mitogen-activated protein kinase kinase 4. The data demonstrate the existence of a novel COP9 signalosome-directed c-Jun activation pathway.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP , MAP Quinasa Quinasa 4 , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Represoras , Factores de Transcripción/metabolismo , Activación Transcripcional , Complejo del Señalosoma COP9 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Eritrocitos/metabolismo , Genes Reporteros , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Quinasas JNK Activadas por Mitógenos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , Péptido Hidrolasas , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/aislamiento & purificación , TransfecciónRESUMEN
Smith-Magenis syndrome (SMS) is a multiple congenital anomaly/mental retardation syndrome associated with an interstitial deletion of chromosome band 17p11.2. The critical region is extremely gene-rich and spans approximately 1.5-2.0 Mb of DNA. Here we report the localization and partial characterization of the gene for subunit 3 of the COP9 signalosome, SGN3. SGN3 maps to the distal portion of the SMS critical interval, between SREBF1 and cCI17-638. We assessed the potential effect of haploinsufficiency of SGN3 in SMS patient lymphoblastoid cell lines through transfection studies and western analysis. Our results indicate that the COP9 signalosome assembles properly in these cells and appears to have normal expression and a kinase function intact. However, because the role of the COP9 signalosome in embryogenesis or differentiation is still uncertain, we cannot rule out the involvement of this gene in the Smith-Magenis syndrome.
Asunto(s)
Anomalías Múltiples/genética , Cromosomas Humanos Par 17/genética , Discapacidad Intelectual/genética , Proteínas Quinasas/genética , Animales , Western Blotting , Células CHO , Complejo del Señalosoma COP9 , Células Cultivadas , Deleción Cromosómica , Mapeo Cromosómico , Cricetinae , ADN/análisis , ADN/genética , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Células Híbridas , Luciferasas/genética , Luciferasas/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Masculino , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Nucleares , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , SíndromeRESUMEN
Polyubiquitin has long been recognised as an intracellular signal. It now appears that different lysine linkages between the ubiquitin moieties are recognised as distinct signals and act in different cell processes. The generation of these different polyubiquitin chains may play an important part in the life of a cell.
Asunto(s)
Biopolímeros/metabolismo , Complejo de la Endopetidasa Proteasomal , Ubiquitinas/metabolismo , Animales , Biopolímeros/química , Biopolímeros/genética , Ligasas/metabolismo , Lisina/química , Mutagénesis Sitio-Dirigida , Péptido Hidrolasas/metabolismo , Poliubiquitina , Transducción de Señal , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Ubiquitinas/química , Ubiquitinas/genéticaRESUMEN
The human core COP9 signalosome consists of eight subunits which have been identified, cloned and sequenced. The components of COP9 signalosome possess homologies with eight non-ATPase regulatory subunits of the 26S proteasome. These polypeptides of the 19S regulator form a reversibly binding subcomplex called the 'lid'. We isolated the 'lid' from human red blood cells and compared it with the COP9 signalosome complex. In addition to the non-ATPase regulatory polypeptides, we found a high molecular mass ATPase copurifying with the human 'lid'. The COP9 signalosome-associated kinase activity is either not at all or only weakly affected by common kinase inhibitors such as 1-(5-Isoquinolinesulfonyl)-2-methyl-piperazine (H7), 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB) or Wortmannin. Curcumin, a tumor suppressor and effector of AP-1 activation, is a potent inhibitor of the COP9 signalosome kinase activity with a Ki of about 10 microM. Since curcumin is known as an inhibitor of the c-Jun N-terminal kinase (JNK) signaling pathway acting upstream of the MAP kinase kinase kinase level, one site of action of the COP9 signalosome might be proximal to regulators on that level.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Transducción de Señal , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , RatonesRESUMEN
We have isolated a fission yeast mutant, mts5-1, in a screen for mutations that confer both methyl 2-benzimidazolecarbamate resistance (MBCR) and temperature sensitivity (ts) on Schizosaccharomyces pombe. This screen has previously isolated mutations in the 26 S proteasome subunits Mts2, Mts3, and Mts4. We show that the mutation in the mts5-1 strain occurs in the pad1(+) gene. pad1(+) was originally isolated on a multicopy plasmid that was capable of conferring staurosporine resistance on a wild type strain. mts5-1/pad1-1 has a similar phenotype to 26 S proteasome mutants previously isolated in the same screen and we show that Pad1 interacts genetically with two of these subunits, Mts3 and Mts4. In this study we describe the identification of Pad1 as a subunit of the 26 S proteasome in fission yeast.
Asunto(s)
Péptido Hidrolasas/genética , Complejo de la Endopetidasa Proteasomal , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario , Proteínas Fúngicas/genética , Genes Fúngicos , Humanos , Ratones , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Schizosaccharomyces/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transactivadores/química , Transactivadores/aislamiento & purificación , Transactivadores/metabolismo , Ubiquitinas/metabolismoRESUMEN
An ATP-dependent protease in the intermembrane space of rat liver mitochondria, MISP I (mitochondrial intermembrane space protease), was partially purified and characterised. The protease complex has a molecular mass of 200 kDa and appears to be an oligomeric enzyme complex. The proteolytic activity of the enzyme can be stimulated up to 3-fold by Mg2+ATP. The Km for ATP is 200 microM. Nucleoside triphosphates, but not ADP, AMP, or nonhydrolysable ATP analogues, can substitute for ATP. The protease exhibits multicatalytic properties with chymotrypsin-like, peptidyl-glutamyl-hydrolysing, and trypsin-like activities. Of the latter the trypsin-like activity is not enhanced by ATP. In addition to the hydrolysis of fluorogenic peptide substrates the protease is able to degrade radiolabeled model proteins. The ATP-dependent mitochondrial protease was characterised as a cysteine protease sensitive to hemine. The cross reactivity of an anti-human-S4 antibody raised against an ATPase subunit of the PA700 complex with a component of MISP I indicated a structural relationship. Furthermore, ATP-agarose-binding assays revealed the connection of the peptide hydrolysing activity with an ATP binding domain. The data presented here and a comparison with known ATP-dependent mitochondrial proteases demonstrated that MISP I represents a novel ATP-dependent protease in the mitochondrial intermembrane space of rat liver.
Asunto(s)
Proteasas ATP-Dependientes , Adenosina Trifosfato/metabolismo , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Mitocondrias Hepáticas/enzimología , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Animales , Endopeptidasas/efectos de los fármacos , Masculino , Mitocondrias Hepáticas/química , Nucleótidos/metabolismo , Inhibidores de Proteasas/farmacología , Ratas , Ratas WistarRESUMEN
A novel protein complex has been identified in human cells that has a molecular mass of approximately 450 kDa. It consists of at least eight different subunits including JAB1, the Jun activation-domain binding protein 1, and Trip15, the thyroid hormone receptor-interacting protein 15. The purified complex contains COP9 and COP11 protein homologs and is very similar, if not identical, to the plant COP9 complex involved in light-mediated signal transduction. The isolated JAB1-containing particle has kinase activity that phosphorylates IkappaBalpha, the carboxy terminus of p105, and Ser63 and/or Ser73 of the amino-terminal activation domain of c-Jun. The phosphorylation of c-Jun requires the carboxy terminus of the protein containing the DNA binding and dimerization domains. Three subunits of the new complex--Sgn3, Sgn5/JAB1, and Sgn6--exhibit sequence similarities to regulatory components of the 26S proteasome, which could indicate the existence of common substrate binding sites. Immunofluorescence staining reveals that the new complex shows a subcellular distribution similar to that of the 26S proteasome. The functional relationship of the two particles in regulating transcriptional activity is discussed. Considering the putative role of the complex in signal transduction and its widespread occurrence, we suggest the name JAB1-containing signalosome.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Complejos Multienzimáticos/metabolismo , Proteínas Quinasas/metabolismo , Proteínas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Sitios de Unión , Complejo del Señalosoma COP9 , Clonación Molecular , Cisteína Endopeptidasas/química , ADN Complementario , Proteínas de Unión al ADN/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Sustancias Macromoleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Proteínas Nucleares , Péptido Hidrolasas , Fosforilación , Complejo de la Endopetidasa Proteasomal , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas/química , Proteínas Proto-Oncogénicas c-jun/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/enzimología , Fracciones Subcelulares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
We have investigated the usefulness of the fission yeast Schizosaccharomyces pombe as a model organism for the discovery of novel modes of drug resistance in human cells. In fission yeast, overexpression of the essential pad1(+) gene confers pleiotropic drug resistance through a pathway involving an AP-1 transcription factor encoded by pap1(+). We have identified POH1, a human pad1 homologue that can substitute fully for pad1(+) and induce AP-1-dependent drug resistance in fission yeast. POH1 also confers P-glycoprotein-independent resistance to taxol (paclitaxel), doxorubicin, 7-hydroxystaurosporine, and ultraviolet light when transiently overexpressed in mammalian cells. Poh1 is a previously unidentified component of the human 26 S proteasome, a multiprotein complex that degrades proteins targeted for destruction by the ubiquitin pathway. Hence, Poh1 is part of a conserved mechanism that determines cellular susceptibility to cytotoxic agents, perhaps by influencing the ubiquitin-dependent proteolysis of transcription factors.
Asunto(s)
Cisteína Endopeptidasas/fisiología , Resistencia a Múltiples Medicamentos , Complejos Multienzimáticos/fisiología , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/genética , Transactivadores/fisiología , Factor de Transcripción AP-1/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Clonación Molecular , Cisteína Endopeptidasas/química , Doxorrubicina/metabolismo , Genes Fúngicos , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Proteínas Asociadas a Pancreatitis , Complejo de la Endopetidasa Proteasomal , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We have isolated a fission yeast gene, mts4(+), by complementation of a temperature-sensitive mutation and show that it encodes subunit 2 (S2) of the 19 S regulatory complex of the 26 S protease. mts4(+) is an essential gene, and we show that loss of this subunit causes cells to arrest in metaphase, illustrating the importance of S2 for mitosis. The Mts4 protein is 48% identical to S2 of the human 26 S protease, and the lethal phenotype of the null mts4 allele can be rescued by the human cDNA encoding S2. We provide genetic and physical evidence to suggest that the Mts4 protein interacts with the product of the mts2(+) gene, an ATPase which has previously been shown to be subunit 4 of the 26 S protease.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Cisteína Endopeptidasas , Endopeptidasas/metabolismo , Mitosis , Péptido Hidrolasas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral , Proteínas Supresoras de Tumor , Alelos , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/genética , Supervivencia Celular , Inhibidor p15 de las Quinasas Dependientes de la Ciclina , Endopeptidasas/química , Endopeptidasas/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Unión Proteica , Conformación Proteica , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Mapeo Restrictivo , Schizosaccharomyces , Factor 2 Asociado a Receptor de TNFRESUMEN
The proteasomal system consists of a proteolytic core, the 20 S proteasome, which associates in an ATP-dependent reaction with the 19 S regulatory complex to form the functional 26 S proteasome. In the absence of ATP, the 20 S proteasome forms a complex with the gamma-interferon-inducible 11 S regulator. Both the 20 S proteasome and the 11 S regulator have been implied in the generation of antigenic peptides. The human immunodeficiency virus (HIV)-1 Tat protein causes a number of different effects during acquired immunodeficiency syndrome (AIDS). Here we show that HIV-1 Tat protein strongly inhibits the peptidase activity of the 20 S proteasome and that it interferes with formation of the 20 S proteasome-11 S regulator complex. In addition, it slightly increases the activity of purified 26 S proteasome. These results may explain the mechanism by which HIV-1-infected cells escape cytotoxic T lymphocyte response and at least in part immunodeficiency in AIDS patients.