RESUMO
We present a combined experimental and theoretical study of beam formation from a cryogenic buffer gas cell. Atoms and molecules are loaded into the cell by laser ablation of a target, and are cooled and swept out of the cell by a flow of cold helium. We study the thermalization and flow dynamics inside the cell and measure how the speed, temperature, divergence and extraction efficiency of the beam are influenced by the helium flow. We use a finite element model to simulate the flow dynamics and use the predictions of this model to interpret our experimental results.
RESUMO
The fluorescence spectrum resulting from laser excitation of the A(2)Π(1/2)âX(2)Σ(+) (0,0) band of ytterbium monofluoride, YbF, has been recorded and analyzed to determine the Franck-Condon factors. The measured values are compared with those predicted from Rydberg-Klein-Rees (RKR) potential energy curves. From the fluorescence decay curve the radiative lifetime of the A(2)Π(1/2) state is measured to be 28 ± 2 ns, and the corresponding transition dipole moment is 4.39 ± 0.16 D. The implications for laser cooling YbF are discussed.
RESUMO
Umbilical defects were induced in a nematic liquid crystal with negative dielectric anisotropy, confined to Hele-Shaw cells with homeotropic boundary conditions, and their annihilation dynamics were investigated experimentally. Dynamic scaling laws, previously proposed for Schlieren defects, were verified also for electric field induced umbilical defects while varying external parameters, such as electric field amplitude, frequency, Hele-Shaw cell gap, and temperature. In all cases, scaling relations of rho(t) proportional to t(-1) for the defect density and D proportional to (t(0) - t)(1/2) for the defect pair separation were obtained, independent of external field parameters. The experimental results give evidence of the universality of scaling relations for the annihilation of topological defects in liquid crystals, extended to umbilical defects and their annihilation dynamics under applied external fields.
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Native disulphide bonds are essential for the structure and function of many membrane and secretory proteins. Disulphide bonds are formed, reduced and isomerized in the endoplasmic reticulum of mammalian cells by a family of oxidoreductases, which includes protein disulphide isomerase (PDI), ERp57, ERp72, P5 and PDIR. This review will discuss how these enzymes are maintained in either an oxidized redox state that allows them to form disulphide bonds in substrate proteins or a reduced form that allows them to perform isomerization and reduction reactions, how these opposing pathways may co-exist within the same compartment and why so many oxidoreductases exist when PDI alone can perform all three of these functions.
Assuntos
Retículo Endoplasmático/metabolismo , Oxigênio/química , Animais , Sítios de Ligação , Dissulfetos/química , Humanos , Oxirredução , Oxigênio/metabolismo , Ligação Proteica , Isomerases de Dissulfetos de Proteínas/química , Dobramento de ProteínaRESUMO
Newly synthesized proteins entering the eukaryotic secretory pathway may be attached to the lipid membrane by essentially one of two mechanisms. They may either contain a hydrophobic stop transfer sequence that directs their integration into the bilayer with the consequence that the polypeptide spans the membrane either one or several times, or alternatively the polypeptide chain may be modified by the covalent addition of a lipid anchor resulting in the attachment of the protein to the membrane via the lipid moiety. The major pathway for the covalent addition of a lipid anchor involves the post-translational attachment of GPI (glycosylphosphatidylinositol) to the C-terminus. Proteins modified in this way contain a specific signal that is recognized by the GPI-anchor processing machinery. Hence both the integration of protein directly into the lipid bilayer and the addition of GPI anchors require the presence of sequences within the polypeptide chain to target the proteins to these pathways. This article will describe the main characteristics of these signals and their similarities and will discuss how the translocon may play a crucial role in their recognition.
Assuntos
Glicosilfosfatidilinositóis/metabolismo , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
Protein folding in living cells is a complex process involving many interdependent factors. The primary site for folding of nascent proteins destined for secretion is the endoplasmic reticulum (ER). Several disease states, including cystic fibrosis, are brought about because of irregularities in protein folding. Under normal cellular conditions, "quality control" mechanisms ensure that only correctly folded proteins are exported from the ER, with incorrectly folded or incompletely assembled proteins being degraded. Quality control mechanisms can be divided into two broad processes: (1) Primary quality control involves general mechanisms that are not specific for individual proteins; these monitor the fidelity of nascent protein folding in the ER and mediate the destruction of incompletely folded proteins. (2) Partially folded or assembled proteins may be subject to secondary quality control mechanisms that are protein- or protein-family-specific. Here we use the folding and assembly of major histocompatibility complex (MHC) class I as an example to illustrate the processes of quality control in the ER. MHC class I, a trimeric complex assembled in the ER of virally infected or malignant cells, presents antigenic peptide to cytotoxic T lymphocytes; this mediates cell killing and thereby prevents the spread of infection or malignancy. The folding and assembly of MHC class I is subjected to both primary and secondary quality control mechanisms that lead either to correct folding, assembly, and secretion or to degradation via a proteasome-associated mechanism.
Assuntos
Retículo Endoplasmático/química , Antígenos de Histocompatibilidade Classe I/química , Animais , Retículo Endoplasmático/imunologia , Humanos , Hidrólise , Dobramento de Proteína , Controle de QualidadeRESUMO
Quality control within the endoplasmic reticulum (ER) is thought to be mediated by the interaction of a folding protein with one or several resident ER proteins [1]. Protein disulphide isomerase (PDI) is one such ER resident protein that has been previously shown to interact with proteins during their folding and assembly pathways [2, 3]. It has been assumed that, as a consequence of this interaction, unassembled proteins are retained within the ER. Here, we experimentally show that this is indeed the case. We have taken advantage of our previous finding that PDI interacts with procollagen chains early on in their assembly pathway [2] to address the role of this protein in directly retaining unassembled chains within the ER. Our experimental approach involved expressing individual C-propeptide domains from different procollagen chains in mammalian cells and determining the ability of these domains to interact with PDI and to be secreted. The C-propeptide from the proalpha2(I) chain was retained within the cell, where it formed a complex with PDI. Conversely, the C-propeptide from the proalpha1(III) chain did not form a complex with PDI and was secreted. Both domains were secreted, however, from a stable cell line expressing a secreted form of PDI lacking its ER retrieval signal. Hence, we have demonstrated directly that the intracellular retention of one substrate for ER quality control is due to an interaction with PDI.
Assuntos
Retículo Endoplasmático/metabolismo , Pró-Colágeno/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Linhagem Celular , Humanos , Oligopeptídeos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Pró-Colágeno/química , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Controle de Qualidade , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.
Assuntos
Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Moléculas de Adesão Celular/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Aciltransferases/metabolismo , Fosfatase Alcalina/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Feminino , Humanos , Células K562 , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Placenta/enzimologia , Gravidez , Biossíntese de Proteínas , Dobramento de Proteína , Subunidades Proteicas , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reticulócitos/metabolismoRESUMO
The presence of correctly formed disulfide bonds is crucial to the structure and function of proteins that are synthesized in the endoplasmic reticulum (ER). Disulfide bond formation occurs in the ER owing to the presence of several specialized catalysts and a suitable redox potential. Work in yeast has indicated that the ER resident glycoprotein Ero1p provides oxidizing equivalents to newly synthesized proteins via protein disulfide isomerase (PDI). Here we show that Ero1-Lalpha, the human homolog of Ero1p, exists as a collection of oxidized and reduced forms and covalently binds PDI. We analyzed Ero1-Lalpha cysteine mutants in the presumed active site C(391)VGCFKC(397). Our results demonstrate that this motif is important for protein folding, structural integrity, protein half-life and the stability of the Ero1-Lalpha-PDI complex.
Assuntos
Glicoproteínas de Membrana/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Dissulfetos/química , Retículo Endoplasmático/química , Glicosilação , Proteína gp120 do Envelope de HIV/química , Células HeLa , Humanos , Oxirredução , Oxirredutases , Conformação Proteica , Dobramento de ProteínaRESUMO
The ability of triple-helical collagen molecules to assemble into supramolecular structures forms the basis of commercial uses of collagen in the food industry and in medical applications such as cosmetic surgery and tissue repair. We have used cDNA techniques to engineer novel collagens with potentially enhanced biological properties; however, expression of fully functional novel molecules is difficult due to the complex nature of procollagen biosynthesis. This article outlines the application of various expression systems to procollagen production and details the use of the mammary gland as a suitable bioreactor for the synthesis of significant amounts of novel procollagens from cDNA constructs.
Assuntos
Biotecnologia/métodos , Colágeno/biossíntese , Colágeno/química , Proteínas Recombinantes/biossíntese , Animais , Linhagem Celular , DNA Complementar/metabolismo , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Pró-Colágeno/biossíntese , Dobramento de Proteína , Proteínas Recombinantes/químicaRESUMO
Oxidative conditions must be generated in the endoplasmic reticulum (ER) to allow disulfide bond formation in secretory proteins. A family of conserved genes, termed ERO for ER oxidoreductins, plays a key role in this process. We have previously described the human gene ERO1-L, which complements several phenotypic traits of the yeast thermo-sensitive mutant ero1-1 (Cabibbo, A., Pagani, M., Fabbri, M., Rocchi, M., Farmery, M. R., Bulleid, N. J., and Sitia, R. (2000) J. Biol. Chem. 275, 4827-4833). Here, we report the cloning and characterization of a novel human member of this family, ERO1-Lbeta. Immunofluorescence, endoglycosidase sensitivity, and in vitro translation/translocation assays reveal that the products of the ERO1-Lbeta gene are primarily localized in the ER of mammalian cells. The ability to allow growth at 37 degrees C and to alleviate the "unfolded protein response" when expressed in ero1-1 cells indicates that ERO1-Lbeta is involved also in generating oxidative conditions in the ER. ERO1-L and ERO1-Lbeta display different tissue distributions. Furthermore, only ERO1-Lbeta transcripts are induced in the course of the unfolded protein response. Our results suggest a complex regulation of ER redox homeostasis in mammalian cells.
Assuntos
Retículo Endoplasmático/enzimologia , Glicoproteínas de Membrana/genética , Oxirredutases/genética , Dobramento de Proteína , Sequência de Aminoácidos , Animais , Compartimento Celular , Biblioteca Gênica , Teste de Complementação Genética , Homeostase , Humanos , Camundongos , Dados de Sequência Molecular , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Hsp47 is a heat-shock protein that interacts transiently with procollagen during its folding, assembly and transport from the endoplasmic reticulum (ER) of mammalian cells. It has been suggested to carry out a diverse range of functions, such as acting as a molecular chaperone facilitating the folding and assembly of procollagen molecules, retaining unfolded molecules within the ER, and assisting the transport of correctly folded molecules from the ER to the Golgi apparatus. Here we define the substrate recognition of Hsp47, demonstrating that it interacts preferentially with triple-helical procollagen molecules. The association of Hsp47 with procollagen coincides with the formation of a collagen triple helix. This demonstrates that Hsp47's role in procollagen folding and assembly is distinct from that of prolyl 4-hydroxylase. These results indicate that Hsp47 acts as a novel molecular chaperone, potentially stabilizing the correctly folded collagen helix from heat denaturation before its transport from the ER.
Assuntos
Proteínas de Choque Térmico/química , Pró-Colágeno/química , Dobramento de Proteína , Animais , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Pró-Colágeno/metabolismo , Ligação Proteica , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
We have established a mammalian semipermeabilized cell system that faithfully reconstitutes the proteasome-mediated degradation of major histocompatibility complex Class I heavy chain. We show that degradation required unfolding of the protein and was cytosol- and ATP-dependent and that dislocation and degradation required proteasome activity. When the interaction of heavy chain with calnexin was prevented, the rate of degradation was accelerated, suggesting that an interaction with calnexin stabilized heavy chain. Stabilization of heavy chain to degradation was also achieved either by preventing mannose trimming or by removal of the N-linked glycosylation site. This demonstrates that glycosylation and mannose trimming are required to ensure degradation of heavy chain. When degradation or mannose trimming was inhibited, heavy chain formed a prolonged interaction with immunoglobulin heavy chain binding protein, ERp57, and protein disulfide isomerase. Taken together, these results indicate that calnexin association and mannose trimming provide a mechanism to regulate the folding, assembly, and degradation of glycoproteins entering the secretory pathway.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Retículo Endoplasmático/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Manose/metabolismo , Substituição de Aminoácidos , Animais , Calnexina , Linhagem Celular , Proteínas de Choque Térmico/metabolismo , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Isomerases/metabolismo , Cinética , Substâncias Macromoleculares , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , Desnaturação Proteica , Isomerases de Dissulfetos de Proteínas/metabolismo , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
We have established a semipermeabilized cell system that reproduces the folding and assembly of a major histocompatibility complex (MHC) class I complex as it would occur in the intact cell. The translation of the MHC class I heavy chain (HLA-B27) in this system was synchronized allowing the folding and assembly of polypeptide chains synthesized within a short time frame to be analyzed. This has enabled us to dissect the time course of interaction of both disulfide and nondisulfide-bonded heavy chain with various molecular chaperones during its assembly in a functionally intact endoplasmic reticulum. The results demonstrate that unassembled, nondisulfide-bonded forms of heavy chain interact initially with calnexin. A later and more prolonged interaction of calreticulin, specifically with assembled, disulfide-bonded heavy chain, highlights distinct differences in the roles of these two proteins in the assembly of MHC class I molecules. We also demonstrate that the thiol-dependent reductase ERp57 initially interacts with nondisulfide-bonded heavy chain, but this rapidly becomes disulfide-bonded and indicates that heavy chain folding occurs during its interaction with ERp57. In addition, we also confirm a direct interaction between MHC class I heavy chain and tapasin, emphasizing the role that this protein plays in the later stages of MHC class I assembly.
Assuntos
Antígeno HLA-B27/genética , Proteínas de Choque Térmico/metabolismo , Isomerases/metabolismo , Biossíntese de Proteínas , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Calreticulina , Linhagem Celular , Permeabilidade da Membrana Celular , Sistema Livre de Células , Clonagem Molecular , Dissulfetos/metabolismo , Genes MHC Classe I , Antígeno HLA-B27/metabolismo , Humanos , Cinética , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas , Coelhos , Proteínas Recombinantes/biossíntese , Reticulócitos/metabolismo , Ribonucleoproteínas/metabolismo , Linfócitos T , Microglobulina beta-2/metabolismoRESUMO
Oxidizing conditions must be maintained in the endoplasmic reticulum (ER) to allow the formation of disulfide bonds in secretory proteins. Here we report the cloning and characterization of a mammalian gene (ERO1-L) that shares extensive homology with the Saccharomyces cerevisiae ERO1 gene, required in yeast for oxidative protein folding. When expressed in mammalian cells, the product of the human ERO1-L gene co-localizes with ER markers and displays Endo-H-sensitive glycans. In isolated microsomes, ERO1-L behaves as a type II integral membrane protein. ERO1-L is able to complement several phenotypic traits of the yeast thermosensitive mutant ero1-1, including temperature and dithiothreitol sensitivity, and intrachain disulfide bond formation in carboxypeptidase Y. ERO1-L is no longer functional when either one of the highly conserved Cys-394 or Cys-397 is mutated. These results strongly suggest that ERO1-L is involved in oxidative ER protein folding in mammalian cells.
Assuntos
Dissulfetos/metabolismo , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Sequência de Aminoácidos , Teste de Complementação Genética , Humanos , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Dados de Sequência Molecular , Oxirredução , Oxirredutases , Oxirredutases atuantes sobre Doadores de Grupo Enxofre , Biossíntese de Proteínas , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Collagen biosynthesis involves a complex series of post-translational modifications, controlled by a number of general and specific molecular chaperones. A recent study has shed new light on the role played in this process by the procollagen-specific chaperone Hsp47.
Assuntos
Colágeno/biossíntese , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Animais , Colágeno/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Choque Térmico/genética , Modelos Biológicos , Transporte ProteicoRESUMO
The correct folding and assembly of proteins within the endoplasmic reticulum (ER) are prerequisites for subsequent transport from this organelle to the Golgi apparatus. The mechanisms underlying the ability of the cell to recognize and retain unassembled or malfolded proteins generally require binding to molecular chaperones within the ER. One classic example of this process occurs during the biosynthesis of procollagen. Here partially folded intermediates are retained and prevented from secretion, leading to a build up of unfolded chains within the cell. The accumulation of these partially folded intermediates occurs during vitamin C deficiency due to incomplete proline hydroxylation, as vitamin C is an essential co-factor of the enzyme prolyl 4-hydroxylase. In this report we show that this retention is tightly regulated with little or no secretion occurring under conditions preventing proline hydroxylation. We studied the molecular mechanism underlying retention by determining which proteins associate with partially folded procollagen intermediates within the ER. By using a combination of cross-linking and sucrose gradient analysis, we show that the major protein binding to procollagen during its biosynthesis is prolyl 4-hydroxylase, and no binding to other ER resident proteins including Hsp47 was detected. This binding is regulated by the folding status rather than the extent of hydroxylation of the chains demonstrating that this enzyme can recognize and retain unfolded procollagen chains and can release these chains for further transport once they have folded correctly.
Assuntos
Retículo Endoplasmático/enzimologia , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Pró-Colágeno/metabolismo , Processamento de Proteína Pós-Traducional , Ácido Ascórbico/farmacologia , Ácido Azetidinocarboxílico/farmacologia , Células Cultivadas , Pró-Colágeno/efeitos dos fármacosRESUMO
We have examined the suitability of the mouse mammary gland for expression of novel recombinant procollagens that can be used for biomedical applications. We generated transgenic mouse lines containing cDNA constructs encoding recombinant procollagen, along with the alpha and beta subunits of prolyl 4-hydroxylase, an enzyme that modifies the collagen into a form that is stable at body temperature. The lines expressed relatively high levels (50-200 micrograms/ml) of recombinant procollagen in milk. As engineered, the recombinant procollagen was shortened and consisted of a pro alpha 2(I) chain capable of forming a triple-helical homotrimer not normally found in nature. Analysis of the product demonstrated that (1) the pro alpha chains formed disulphide-linked trimers, (2) the trimers contained a thermostable triple-helical domain, (3) the N-propeptides were aligned correctly, and (4) the expressed procollagen was not proteolytically processed to collagen in milk.
Assuntos
Glândulas Mamárias Animais/metabolismo , Leite/metabolismo , Pró-Colágeno/biossíntese , Engenharia de Proteínas , Animais , Northern Blotting , Southern Blotting , Feminino , Camundongos , Camundongos Transgênicos , Pró-Colágeno/química , Pró-Colágeno/genética , Pró-Colágeno-Prolina Dioxigenase/genética , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/químicaRESUMO
Schmid metaphyseal chondrodysplasia results from mutations within the COOH-terminal globular domain (NC1) of type X collagen, a short chain collagen expressed in the hypertrophic region of the growth plate cartilage. Previous in vitro studies have proposed that mutations prevent the association of the NC1 domain of constituent chains of the trimer based upon a lack of formation of a trimeric structure that is resistant to dissociation with sodium dodecyl sulfate. To examine the effect of mutations on folding and assembly within a cellular context, bovine type X cDNAs containing analogous disease causing mutations Y598D, N617K, W651R, and wild-type were expressed in semi-permeabilized cells. We assessed trimerization of the mutant chains by their ability to form a collagen triple helix. Using this approach, we demonstrate that although there is an apparent lower efficiency of association of the mutant NC1 domains, they can drive the formation of correctly aligned triple helices with the same thermal stability as the wild-type collagen. When epitope-tagged mutant and wild-type collagen were co-expressed, heterotrimers could be detected by sequential immunoprecipitation. Both wild-type and mutant type X chains were found in association with the molecular chaperones protein disulfide isomerase and Hsp 47. The implications of these findings on the likely mechanism of Schmid metaphyseal chondrodysplasia will be discussed.