RESUMEN
During embryo development, human primordial germ cells (hPGCs) express a naive gene expression program with similarities to pre-implantation naive epiblast (EPI) cells and naive human embryonic stem cells (hESCs). Previous studies have shown that TFAP2C is required for establishing naive gene expression in these cell types, however the role of additional naive transcription factors in hPGC biology is not known. Here, we show that unlike TFAP2C, the naive transcription factors KLF4 and TFCP2L1 are not required for induction of hPGC-like cells (hPGCLCs) from hESCs, and they have no role in establishing and maintaining a naive-like gene expression program in hPGCLCs with extended time in culture. Taken together, our results suggest a model whereby the molecular mechanisms that drive naive gene expression in hPGCs/hPGCLCs are distinct from those in the naive EPI/hESCs.
Asunto(s)
Células Germinativas , Células Madre Embrionarias Humanas , Diferenciación Celular , Células Germinativas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Factor 4 Similar a Kruppel , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , TranscriptomaRESUMEN
The generation of patient-specific pluripotent stem cells has the potential to accelerate the implementation of stem cells for clinical treatment of degenerative diseases. Technologies including somatic cell nuclear transfer and cell fusion might generate such cells but are hindered by issues that might prevent them from being used clinically. Here, we describe methods to use dermal fibroblasts easily obtained from an individual human to generate human induced pluripotent stem (iPS) cells by ectopic expression of the defined transcription factors KLF4, OCT4, SOX2, and C-MYC. The resultant cell lines are morphologically indistinguishable from human embryonic stem cells (HESC) generated from the inner cell mass of a human preimplantation embryo. Consistent with these observations, human iPS cells share a nearly identical gene-expression profile with two established HESC lines. Importantly, DNA fingerprinting indicates that the human iPS cells were derived from the donor material and are not a result of contamination. Karyotypic analyses demonstrate that reprogramming of human cells by defined factors does not induce, or require, chromosomal abnormalities. Finally, we provide evidence that human iPS cells can be induced to differentiate along lineages representative of the three embryonic germ layers indicating the pluripotency of these cells. Our findings are an important step toward manipulating somatic human cells to generate an unlimited supply of patient-specific pluripotent stem cells. In the future, the use of defined factors to change cell fate may be the key to routine nuclear reprogramming of human somatic cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Dermis/citología , Fibroblastos/citología , Células Madre Pluripotentes/citología , Ingeniería de Tejidos/métodos , Factores de Transcripción/metabolismo , Diferenciación Celular/fisiología , ADN Complementario/genética , Fibroblastos/metabolismo , Fibroblastos/fisiología , Perfilación de la Expresión Génica , Vectores Genéticos/genética , Humanos , Factor 4 Similar a Kruppel , Análisis por Micromatrices , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiologíaRESUMEN
BACKGROUND: Basophils participate in allergic diseases by invading affected tissues and secreting histamine, leukotriene (LT)C4, IL-4 and IL-13 following FcepsilonRI cross-linking. A reduction of basophil mediator production is therefore of considerable therapeutical interest. Macrolactam derivatives, which inhibit calcineurin activation, may be candidates for antiallergic therapy as they reduce both symptoms of inflammatory skin disease in animal models and mast cell degranulation. OBJECTIVE: To investigate the effects of the calcineurin antagonists ascomycin and cyclosporin A on IgE-dependent mediator release from human basophils. METHODS: Basophils were purified by Ficoll density centrifugation, elutriation and negative selection. Histamine release was measured spectrofluorometrically; LTC4, IL-4 and IL-13 secretions were assayed by enzyme-linked immunosorbent assay (ELISA). Lysed cells were subjected to Western blotting using specific antibodies to phospho-p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)-1 and -2. RESULTS: Ascomycin (0.01 nm to 1 micro m) and cyclosporin A (0.1 nm to 10 micro m) strikingly inhibited (maximally 100%) anti-IgE-induced histamine and cytokine release from basophils, and these actions were unaffected by IL-3 priming. Ascomycin, however, was less potent at blocking LTC4 secretion, whereas cyclosporin A was unable to block production of this mediator. In immunoblotting studies, ascomycin and cyclosporin A reduced IgE-dependent p38 MAPK activation but were less potent at reducing ERK phosphorylation in basophils. CONCLUSION: Calcineurin antagonists like ascomycin and cyclosporin A block IgE-dependent basophil degranulation and cytokine synthesis. Calcineurin may target p38 MAPK activation, but seems to have less activity on ERK phosphorylation. This is paralleled by a reduced or even absent effect of calcineurin antagonists on eicosanoid production.
Asunto(s)
Basófilos/metabolismo , Ciclosporina/farmacología , Inmunosupresores/farmacología , Interleucinas/metabolismo , Leucotrieno C4/metabolismo , Tacrolimus/análogos & derivados , Basófilos/inmunología , Western Blotting , Inhibidores de la Calcineurina , Ensayo de Inmunoadsorción Enzimática , Liberación de Histamina/efectos de los fármacos , Liberación de Histamina/fisiología , Humanos , Tacrolimus/farmacologíaRESUMEN
Mercuric chloride (HgCl2) is an industrial agent known to cause autoimmune disorders and induce IgE synthesis, which plays a crucial role in the manifestation of allergic diseases. In rodents, the immunomodulatory effects of HgCl2 have been shown to involve the enhancement of mast cell-derived IL-4 secretion, which facilitates both Th2-lymphocyte development and IgE production. In humans, rapid allergen-dependent release of IL-4 and the related cytokine IL-13 from histamine-containing cells occurs primarily in basophils, along with other proinflammatory mediators such as histamine and LTC4. In this study, we therefore investigated the effects of HgCl2 on the release of the above basophil mediators, either due to the compound alone or in conjunction with IgE-dependent stimulation. HgCl2 (10(-9) to 10(-6) M) did not induce mediator secretion alone but significantly enhanced the release of histamine, LTC4, IL-4, and IL-13 caused by anti-IgE. Higher concentrations of HgCl2 (10(-5) to 10(-3) M) strikingly reduced cell viability; however, toxicity varied depending on cell density and incubation time. Removal of HgCl2 following a short incubation with basophils did not reverse the potentiating effects on basophil mediator secretion to anti-IgE and the concentration of free mercury in the supernatants significantly diminished by up to 20% after incubation with the cells, indicating irreversible Hg binding to cells. By upregulating IgE-dependent human basophil mediator release, our results clearly indicate that HgCl2 potentially exacerbates allergic disorders and promotes a Th2-cytokine profile.
Asunto(s)
Basófilos/efectos de los fármacos , Inmunoglobulina E/inmunología , Cloruro de Mercurio/farmacología , Anticuerpos Antiidiotipos/farmacología , Basófilos/inmunología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Liberación de Histamina/efectos de los fármacos , Humanos , Interleucina-13/análisis , Interleucina-4/análisis , Leucotrieno C4/análisis , Cloruro de Mercurio/toxicidadAsunto(s)
Basófilos/fisiología , Canales de Cloruro/antagonistas & inhibidores , Liberación de Histamina/efectos de los fármacos , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Basófilos/efectos de los fármacos , Bumetanida/farmacología , Canales de Cloruro/fisiología , Diuréticos/farmacología , Furosemida/farmacología , Humanos , Inmunoglobulina E/fisiologíaRESUMEN
Cotranslational translocation of proteins requires ribosome binding to the Sec61p channel at the endoplasmic reticulum (ER) membrane. We have used electron cryomicroscopy to determine the structures of ribosome-channel complexes in the absence or presence of translocating polypeptide chains. Surprisingly, the structures are similar and contain 3-4 connections between the ribosome and channel that leave a lateral opening into the cytosol. Therefore, the ribosome-channel junction may allow the direct transfer of polypeptides into the channel and provide a path for the egress of some nascent chains into the cytosol. Moreover, complexes solubilized from mammalian ER membranes contain an additional membrane protein that has a large, lumenal protrusion and is intercalated into the wall of the Sec61p channel. Thus, the native channel contains a component that is not essential for translocation.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Extensión de la Cadena Peptídica de Translación , Ribosomas/química , Ribosomas/metabolismo , Animales , Microscopía por Crioelectrón , Citoplasma/metabolismo , Perros , Retículo Endoplásmico/química , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Sustancias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Conejos , Canales de Translocación SEC , Levaduras/química , Levaduras/citología , Levaduras/metabolismoRESUMEN
In posttranslational translocation in yeast, completed protein substrates are transported across the endoplasmic reticulum membrane through a translocation channel formed by the Sec complex. We have used photo-cross-linking to investigate interactions of cytosolic proteins with a substrate synthesized in a reticulocyte lysate system, before its posttranslational translocation through the channel in the yeast membrane. Upon termination of translation, the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC) are released from the polypeptide chain, and the full-length substrate interacts with several different cytosolic proteins. At least two distinct complexes exist that contain among other proteins either 70-kD heat shock protein (Hsp70) or tailless complex polypeptide 1 (TCP1) ring complex/chaperonin containing TCP1 (TRiC/CCT), which keep the substrate competent for translocation. None of the cytosolic factors appear to interact specifically with the signal sequence. Dissociation of the cytosolic proteins from the substrate is accelerated to the same extent by the Sec complex and an unspecific GroEL trap, indicating that release occurs spontaneously without the Sec complex playing an active role. Once bound to the Sec complex, the substrate is stripped of all cytosolic proteins, allowing it to subsequently be transported through the membrane channel without the interference of cytosolic binding partners.
Asunto(s)
Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae , Chaperonina con TCP-1 , Chaperoninas/metabolismo , Reactivos de Enlaces Cruzados , Proteínas Fúngicas/metabolismo , Modelos Biológicos , Chaperonas Moleculares , Precursores de Proteínas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Transactivadores/metabolismoRESUMEN
Posttranslational protein translocation across the membrane of the endoplasmic reticulum is mediated by the Sec complex. This complex includes a transmembrane channel formed by multiple copies of the Sec61 protein. Translocation of a polypeptide begins when the signal sequence binds at a specific site within the channel. Binding results in the insertion of the substrate into the channel, possibly as a loop with a small segment exposed to the lumen. While bound, the signal sequence is in contact with both protein components of the channel and the lipid of the membrane. Subsequent movement of the polypeptide through the channel occurs when BiP molecules interact transiently with a luminal domain of the Sec complex, hydrolyze ATP, and bind to the substrate. Bound BiP promotes translocation by preventing the substrate from diffusing backwards through the channel, and thus acts as a molecular ratchet.
Asunto(s)
Retículo Endoplásmico/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Humanos , Membranas Intracelulares/metabolismo , Mitocondrias/metabolismo , Señales de Clasificación de Proteína/metabolismoRESUMEN
We have addressed the mechanism by which proteins are posttranslationally transported across the membrane of the yeast endoplasmic reticulum (ER). We demonstrate that BiP (Kar2p), a member of the Hsp70 family resident in the ER lumen, acts as a molecular ratchet during translocation of the secretory protein prepro-alpha factor through the channel formed by the Sec complex. Multiple BiP molecules associate with each translocation substrate following interaction with the J domain of the Sec63p component of the Sec complex. Bound BiP minimizes passive backward movements of the substrate through the channel, and BiP's subsequent dissociation results in a free polypeptide in the ER lumen. Antibodies against the substrate can replace BiP, indicating that a Brownian ratchet is sufficient to achieve translocation.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli , Proteínas Fúngicas/química , Proteínas HSP70 de Choque Térmico/química , Membranas Intracelulares/metabolismo , Cinética , Factor de Apareamiento , Datos de Secuencia Molecular , Péptidos/genética , Unión Proteica , Biosíntesis de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
We have analyzed how the signal sequence of prepro-alpha-factor is recognized during the first step of posttranslational protein transport into the yeast endoplasmic reticulum. Cross-linking studies indicate that the signal sequence interacts in a Kar2p- and ATP-independent reaction with Sec61p, the multispanning membrane component of the protein-conducting channel, by intercalation into transmembrane domains 2 and 7. While bound to Sec61p, the signal sequence forms a helix that is contacted on one side by Sec62p and Sec71p. The binding site is located at the interface of the protein channel and the lipid bilayer. Signal sequence recognition in cotranslational translocation in mammals appears to occur similarly. These results suggest a general mechanism by which the signal sequence could open the channel for polypeptide transport.
Asunto(s)
Retículo Endoplásmico/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Señales de Clasificación de Proteína/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico/fisiología , Reactivos de Enlaces Cruzados/metabolismo , Retículo Endoplásmico/química , Proteínas Fúngicas/análisis , Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Lisina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Mutagénesis/fisiología , Fenilalanina/análogos & derivados , Fotoquímica , Señales de Clasificación de Proteína/análisis , Señales de Clasificación de Proteína/genética , Canales de Translocación SEC , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMEN
Posttranslational protein translocation across the endoplasmic reticulum membrane of yeast requires a seven-component transmembrane complex (the Sec complex) in collaboration with the lumenal Kar2 protein (Kar2p). A translocation substrate was initially bound to the cytosolic face of the purified Sec complex in a signal-sequence-dependent but Kar2p- and nucleotide-independent manner. In a subsequent reaction, in which Kar2p interacted with the lumenal face of the Sec complex and hydrolyzed adenosine triphosphate, the substrate moved through a channel formed by the Sec complex and was released at the lumenal end. Movement through the channel occurred in detergent solution in the absence of a lipid bilayer.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfato/metabolismo , Transporte Biológico , Reactivos de Enlaces Cruzados , Citosol/metabolismo , Detergentes , Digitonina , Retículo Endoplásmico/metabolismo , Membrana Dobles de Lípidos , Liposomas/metabolismo , Señales de Clasificación de Proteína/metabolismo , Proteolípidos/metabolismo , ARN de Transferencia/metabolismo , Canales de Translocación SEC , Solubilidad , SuccinimidasRESUMEN
The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.
Asunto(s)
Retículo Endoplásmico/ultraestructura , Proteínas de Choque Térmico , Canales Iónicos/ultraestructura , Proteínas de la Membrana/ultraestructura , Proteínas de Transporte de Membrana , Proteolípidos/ultraestructura , Proteínas de Saccharomyces cerevisiae , Animales , Transporte Biológico , Compartimento Celular , Detergentes , Perros , Técnica de Fractura por Congelación , Proteínas Fúngicas/metabolismo , Aumento de la Imagen , Activación del Canal Iónico , Sustancias Macromoleculares , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Peso Molecular , Movimiento (Física) , Coloración Negativa , Tamaño de la Partícula , Unión Proteica , Biosíntesis de Proteínas , Conformación Proteica , Ribosomas/metabolismo , Canales de Translocación SEC , LevadurasRESUMEN
Yeast microsomes contain a heptameric Sec complex involved in post-translational protein transport that is composed of a heterotrimeric Sec61p complex and a tetrameric Sec62-Sec63 complex. The trimeric Sec61p complex also exists as a separate entity that probably functions in co-translational protein transport, like its homolog in mammals. We have now discovered in the yeast endoplasmic reticulum membrane a second, structurally related trimeric complex, named Ssh1p complex. It consists of Ssh1p1 (Sec sixty-one homolog 1), a rather distant relative of Sec61p, of Sbh2p, a homolog of the Sbh1p subunit of the Sec61p complex, and of Sss1p, a component common to both trimeric complexes. In contrast to Sec61p, Ssh1p is not essential for cell viability but it is required for normal growth rates. Sbh1p and Sbh2p individually are also not essential, but cells lacking both proteins are impaired in their growth at elevated temperatures and accumulate precursors of secretory proteins; microsomes isolated from these cells also exhibit a reduced rate of post-translational protein transport. Like the Sec61p complex, the Ssh1p complex interacts with membrane-bound ribosomes, but it does not associate with the Sec62-Sec63p complex to form a heptameric Sec complex. We therefore propose that it functions exclusively in the co-translational pathway of protein transport.
Asunto(s)
Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico Activo , ADN de Hongos/genética , Perros , Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/genética , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Procesamiento Proteico-Postraduccional , Canales de Translocación SEC , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Homología de Secuencia de AminoácidoRESUMEN
A recently described downstream target of mitogen-activated protein kinases (MAPKs) is the MAPK-activated protein (MAPKAP) kinase 2 which has been shown to be responsible for small heat shock protein phosphorylation. We have analyzed the mechanism of MAPKAP kinase 2 activation by MAPK phosphorylation using a recombinant MAPKAP kinase 2-fusion protein, p44MAPK and p38/40MAPK in vitro and using an epitope-tagged MAPKAP kinase 2 in heat-shocked NIH 3T3 cells. It is demonstrated that, in addition to the known phosphorylation of the threonine residue carboxyl-terminal to the catalytic domain, Thr-317, activation of MAPKAP kinase 2 in vitro and in vivo is dependent on phosphorylation of a second threonine residue, Thr-205, which is located within the catalytic domain and which is highly conserved in several protein kinases. Constitutive activation of MAPKAP kinase 2 is obtained by replacement of both of these threonine residues by glutamic acid. A constitutively active form of MAPKAP kinase 2 is also obtained by deletion of a carboxyl-terminal region containing Thr-317 and the A-helix motif or by replacing the conserved residues of the A-helix. These data suggest a dual mechanism of MAPKAP kinase 2 activation by phosphorylation of Thr-205 inside the catalytic domain and by phosphorylation of Thr-317 outside the catalytic domain involving an autoinhibitory A-helix motif.
Asunto(s)
Proteínas Quinasas Activadas por Mitógenos , Mutación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Cartilla de ADN/genética , Activación Enzimática , Calor , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Ratones , Proteína Quinasa 3 Activada por Mitógenos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Células Tumorales CultivadasRESUMEN
The primary structure of mouse MAP kinase-activated protein (MAPKAP) kinase 2 contains a proline-rich N-terminal region which might function as a src-homology 3 (SH3) domain-binding motif in vivo. To demonstrate the ability of this region to bind SH3 domains, we analyzed the interaction of the SH3 domain of the protein tyrosine kinase c-abl with MAPKAP kinase 2. It is demonstrated, that the proline-rich region specifically binds c-abl-SH3 domain in vitro. Furthermore, it is shown, that deletion of this proline-rich region does not significantly influence the substrate binding properties of the enzyme when analyzed with the substrate small heat shock protein Hsp25. The data suggest that the proline-rich region of MAPKAP kinase 2 could interact with proteins containing SH3-domains also in vivo regulating its cellular localization and/or modulating its enzymatic properties.
Asunto(s)
Prolina , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catálisis , ADN Complementario/química , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Datos de Secuencia Molecular , Relación Estructura-ActividadRESUMEN
The protein sequence of MAP kinase-activated protein kinase 2 (MAPKAP kinase 2) deduced from mouse cDNA sequence reveals structural features of the enzyme, which could be of importance for its function: a proline-rich SH3-binding domain N-terminal to the catalytic region, a MAP kinase phosphorylation site and a bipartite nuclear targeting sequence located C-terminal to the catalytic region. The catalytic domain itself has the strongest homology to calcium/calmodulin-dependent protein kinase II. Northern blot analysis demonstrates a 3.5 kb MAPKAP kinase 2 transcript which is ubiquitously expressed and, hence, co-expressed with the mRNA of the recently identified substrate Hsp25 in all tissues analysed. However, the functional consequences of the nuclear targeting sequence present in MAPKAP kinase 2 suggest the existence of further substrates for the enzyme in the nucleus.