RESUMEN
Disulphide bonds are critical for the maturation and stability of secretory and cell-surface proteins. In eukaryotic cells, disulphide bonds are introduced in the ER (endoplasmic reticulum), where the redox conditions are optimal to support their formation. Yet, the correct pairing of cysteine residues is not simple and often requires the assistance of redox-active proteins. The enzymes of the thiol-disulphide oxidoreductase family catalyse oxidation, reduction and isomerization, and thereby play important roles for the folding of many proteins. To allow all three redox reactions to take place concurrently in the same compartment, specific protein-protein interactions regulate the function of individual enzymes, while a careful balance of the ER redox environment is maintained. At the same time, the system must be capable of responding to changes in the cellular conditions, caused, for instance, by oxidative stress and protein misfolding. This review presents recent progress in understanding how ER redox conditions are regulated and how protein disulphides are formed in the ER of mammalian cells.
Asunto(s)
Disulfuros , Retículo Endoplásmico/metabolismo , Animales , Sitios de Unión , Catálisis , Disulfuros/química , Humanos , Modelos Biológicos , Modelos Moleculares , Oxidación-Reducción , Estrés Oxidativo , Oxígeno/química , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismoRESUMEN
The process of 'quality control' in the endoplasmic reticulum (ER) involves a variety of mechanisms that collectively ensure that only correctly folded, assembled and modified proteins are transported along the secretory pathway. In contrast, non-native proteins are retained and eventually targeted for degradation. Recent work provides the first structural insights into the process of glycoprotein folding in the ER involving the lectin chaperones calnexin and calreticulin. Underlying principles governing the choice of chaperone system engaged by different proteins have also been discovered.
Asunto(s)
Retículo Endoplásmico/fisiología , Chaperonas Moleculares/química , Animales , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calnexina , Calreticulina , Glucosiltransferasas/metabolismo , Glicoproteínas/química , Glicoproteínas/metabolismo , Proteínas de Choque Térmico/metabolismo , Isomerasas/metabolismo , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismoRESUMEN
The NMR structure of the rat calreticulin P-domain, comprising residues 189-288, CRT(189-288), shows a hairpin fold that involves the entire polypeptide chain, has the two chain ends in close spatial proximity, and does not fold back on itself. This globally extended structure is stabilized by three antiparallel beta-sheets, with the beta-strands comprising the residues 189-192 and 276-279, 206-209 and 262-265, and 223-226 and 248-251, respectively. The hairpin loop of residues 227-247 and the two connecting regions between the beta-sheets contain a hydrophobic cluster, where each of the three clusters includes two highly conserved tryptophyl residues, one from each strand of the hairpin. The three beta-sheets and the three hydrophobic clusters form a repeating pattern of interactions across the hairpin that reflects the periodicity of the amino acid sequence, which consists of three 17-residue repeats followed by three 14-residue repeats. Within the global hairpin fold there are two well-ordered subdomains comprising the residues 219-258, and 189-209 and 262-284, respectively. These are separated by a poorly ordered linker region, so that the relative orientation of the two subdomains cannot be precisely described. The structure type observed for CRT(189-288) provides an additional basis for functional studies of the abundant endoplasmic reticulum chaperone calreticulin.
Asunto(s)
Proteínas de Unión al Calcio/química , Ribonucleoproteínas/química , Secuencia de Aminoácidos , Animales , Calreticulina , Simulación por Computador , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Estructura Terciaria de Proteína , RatasRESUMEN
Calreticulin (CRT) is an abundant molecular chaperone of the endoplasmic reticulum. Its central, proline-rich P-domain, comprising residues 189-288, contains three copies of each of two repeat sequences (types 1 and 2), which are arranged in a characteristic '111222' pattern. Here we show that the three-dimensional structure of CRT(189-288) contains a single hairpin fold formed by the entire polypeptide chain. The loop at the bottom of the hairpin consists of residues 227-247, and is closed by an anti-parallel beta-sheet of residues 224-226 and 248-250. Two additional beta-sheets contain residues 207-209 and 262-264, and 190-192 and 276-278. The 17-residue spacing of the beta-strands in the N-terminal part of the hairpin and the 14-residue spacing in the C-terminal part reflect the length of the type 1 and type 2 sequence repeats. As a consequence of this topology the peptide segments separating the beta-strands in the N-terminal part of the hairpin are likely to form bulges to accommodate the extra residues. These results are based on nearly complete sequence-specific NMR assignments for CRT(189-288), which were obtained using standard NMR techniques with the (13)C/(15)N-labeled protein, and collection of nuclear Overhauser enhancement upper distance constraints.
Asunto(s)
Proteínas de Unión al Calcio/química , Fragmentos de Péptidos/química , Ribonucleoproteínas/química , Secuencia de Aminoácidos , Animales , Calreticulina , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Prolina/análisis , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Secuencias Repetitivas de Aminoácido , SolucionesRESUMEN
A variety of quality control mechanisms operate in the endoplasmic reticulum and in downstream compartments of the secretory pathway to ensure the fidelity and regulation of protein expression during cell life and differentiation. As a rule, only proteins that pass a stringent selection process are transported to their target organelles and compartments. If proper maturation fails, the aberrant products are degraded. Quality control improves folding efficiency by retaining proteins in the special folding environment of the endoplasmic reticulum, and it prevents harmful effects that could be caused by the deployment of incompletely folded or assembled proteins.
Asunto(s)
Retículo Endoplásmico/metabolismo , Pliegue de Proteína , Proteínas/química , Proteínas/metabolismo , Animales , Glicoproteínas/química , Glicoproteínas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Oligosacáridos/metabolismo , Orgánulos/metabolismo , Conformación ProteicaRESUMEN
The 39-kDa receptor-associated protein (RAP) is an intracellular glycoprotein that interacts with hitherto unknown sites in several members of the low-density-lipoprotein receptor gene family. Upon binding to these receptors, RAP inhibits all ligand interactions with the receptors. In the present study, the transglutaminase-catalyzed incorporation of radioactively labeled putrescine and a dansylated glutamine-containing peptide into human RAP has been studied. The results indicate the presence of both glutamine and lysine residues in RAP, accessible for transglutaminase cross-linking. Moreover, enzymatic digestion followed by sequence analysis of radiolabeled fractions demonstrated that Gln261 acts as the amine acceptor site. This residue is located in the third domain of RAP and is conserved among the RAP interspecies homologues. Insertion of a reporter group into the protein could prove useful to assess ligand/receptor interactions.
Asunto(s)
Proteínas Portadoras/química , Glutamina/química , Glicoproteínas/química , Transglutaminasas/química , Animales , Electroforesis en Gel de Poliacrilamida , Genes Reporteros , Cobayas , Humanos , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Hígado/enzimología , Espectroscopía de Resonancia Magnética , Placenta/metabolismo , Putrescina/metabolismo , Factores de TiempoRESUMEN
The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family.
Asunto(s)
Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Ligandos , Receptores de LDL/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Unión Competitiva/genética , Células CHO , Proteínas del Sistema Complemento/genética , Cricetinae , Reactivos de Enlaces Cruzados/metabolismo , Glutaral/metabolismo , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Unión Proteica , ARN Mensajero/análisis , Receptores de LDL/genética , Serina Endopeptidasas/metabolismo , Serpinas/metabolismo , TransfecciónRESUMEN
Binding of the receptor-associated protein (RAP) to the newly identified putative sorting receptor, sortilin, was analyzed by surface plasmon resonance analysis of recombinant RAP and sortilin domains and compared with binding to megalin and low density lipoprotein receptor-related protein (LRP). The data show that the RAP-binding site in sortilin is localized in the cysteine-rich lumenal part homologous to yeast vacuolar protein-sorting 10 protein (Vps10p), and the sortilin-binding site in RAP is localized in the carboxy-terminal domain III of the three homologous domains in RAP. Whereas sortilin bound only RAP domain III, megalin and LRP bound all RAP domains with the functional affinity order: domain III >domain I > domain II.
Asunto(s)
Proteínas Fúngicas/química , Glicoproteínas de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Proteínas Adaptadoras del Transporte Vesicular , Animales , Células CHO/metabolismo , Cricetinae , Complejo Antigénico de Nefritis de Heymann , Glicoproteínas de Membrana/química , Proteínas del Tejido Nervioso/química , Conformación Proteica , Receptores de LDL/metabolismoRESUMEN
The three-dimensional structure of the N-terminal domain (residues 18-112) of alpha2-macroglobulin receptor-associated protein (RAP) has been determined by NMR spectroscopy. The structure consists of three helices composed of residues 23-34, 39-65, and 73-88. The three helices are arranged in an up-down-up antiparallel topology. The C-terminal 20 residues were shown not to be in a well defined conformation. A structural model for the binding of RAP to the family of low-density lipoprotein receptors is proposed. It defines a role in binding for both the unordered C terminus and the structural scaffold of the core structure. Pathogenic epitopes for the rat disease Heymann nephritis, an experimental model of human membranous glomerulonephritis, have been identified in RAP and in the large endocytic receptor gp330/megalin. Here we provide the three-dimensional structure of the pathogenic epitope in RAP. The amino acid residues known to form the epitope are in a helix-loop-helix conformation, and from the structure it is possible to rationalize the published results obtained from studies of fragments of the N-terminal domain.
Asunto(s)
Proteínas Portadoras/química , Glicoproteínas/química , Animales , Proteínas Portadoras/inmunología , Mapeo Epitopo , Escherichia coli , Glicoproteínas/inmunología , Humanos , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Datos de Secuencia Molecular , Mapeo Peptídico , Conformación Proteica , RatasRESUMEN
The alpha2macroglobulin-receptor-associated protein (RAP) binds to the alpha2macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha2MR/LRP), a multi-functional cell surface receptor known to bind and internalize several macromolecular ligands. RAP has been shown to inhibit binding of all known alpha2MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands. In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously [Warshawsky, I., Bu, G. & Schwartz, A. L. (1995) Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein, Biochemistry 34, 3404-3415], to three structural domains, 1, 2 and 3, comprising residues 18-112, 113-218 and 219-323 of RAP, respectively. Structural analysis by 1H-NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of alpha-helices, whereas domain 3 as a separate domain appears only to be marginally stable. Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1+2 and 2+3 against 125I-RAP and 125I-alpha2M* (methylamine-activated alpha2M) for binding to alpha2MR/LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C-terminal regions of domains 1 and 3.
Asunto(s)
Proteínas Portadoras/química , Glicoproteínas/química , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cartilla de ADN/genética , Escherichia coli/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Ligandos , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Estructura Secundaria de Proteína , Receptores Inmunológicos/metabolismo , Receptores de LDL/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , alfa-Macroglobulinas/metabolismoRESUMEN
The receptor-associated protein, RAP, is an intracellular protein that may function as a chaperone for the LDL-receptor family receptors. Here we report calmodulin as the first identified RAP binding protein outside of the LDL-receptor family members. We demonstrate that RAP binds calmodulin in a Ca2+- and pH-dependent manner characteristic of calmodulin-dependent enzymes, and present evidence that RAP is a substrate for calmodulin-dependent enzymes. Thus, CaM-kinase II and calcineurin readily phosphorylate and dephosphorylate, respectively, serine residues in RAP, and in the individual RAP domains D2 (amino acids 113-218) and D3 (amino acids 219-323) which both contain sites for CaM-kinase II-mediated phosphorylation and for calmodulin binding. In addition, we provide evidence that RAP is phosphorylated by other kinases such as casein kinase II. Studies of 32[ortho]P-labelled cell cultures demonstrate that RAP is phosphorylated in vivo. Our results suggest that RAP may have hitherto unknown functions implicating phosphorylation and calmodulin-mediated modulation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Proteínas Portadoras/metabolismo , Glicoproteínas/metabolismo , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Calcineurina , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas de Unión a Calmodulina/metabolismo , Quinasa de la Caseína II , Concentración de Iones de Hidrógeno , Proteína Asociada a Proteínas Relacionadas con Receptor de LDL , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Fosfoserina/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de LDL/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Very low density lipoprotein receptor (VLDL-R) was found to be expressed in bovine mammary gland and the human breast carcinoma cell line MCF-7 as an M(r) 105,000 variant, and in Chinese hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M(r) 130,000 variant. The receptor was purified by ligand affinity chromatography with immobilized M(r) 40,000 receptor-associated protein (RAP). The purified receptor was found to bind urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex (u-PA.PAI-1), while there was no or very weak binding of active site blocked u-PA (DFP-u-PA), PAI-1 or u-PA-type-2 plasminogen activator inhibitor complex. The binding of u-PA.PAI-1 was blocked by RAP. The transfected CHO cells had an efficient, RAP-sensitive endocytosis of u-PA.PAI-1, severalfold higher than non-transfected parental CHO cells. u-PA.PAI-1 endocytosis was partially inhibited by DFP-u-PA, which blocks binding of the complex to the u-PA receptor. RAP and DFP-u-PA sensitive u-PA.PAI-1 endocytosis was also observed in MCF-7 cells, which were without detectable levels of other RAP-binding endocytosis receptors. These results show that VLDL-R represents a novel endocytosis mechanism for u-PA receptor-bound u-PA.PAI-1.
Asunto(s)
Endocitosis , Glándulas Mamarias Animales/metabolismo , Inhibidor 1 de Activador Plasminogénico/metabolismo , Receptores de LDL/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Neoplasias de la Mama , Células CHO , Bovinos , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Variación Genética , Humanos , Cinética , Lipoproteínas VLDL/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Péptidos/síntesis química , Péptidos/inmunología , Receptores de LDL/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Transfección , Células Tumorales CultivadasRESUMEN
Complex between urokinase and its type-1 inhibitor (uPA-PAI-1) may, when bound to the urokinase receptor (uPAR), be endocytosed by an ensuing binding of the complex to the multiligand receptors alpha (2)-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and glycoprotein 330 (gp330). We have found that phorbol esters regulate endocytosis of uPA-PAI-1 differently in different cell lines. In COS-1 cells, expressing uPAR and high levels of alpha 2MR/LRP under basal conditions, phorbol esters cause a time-dependent decrease in endocytosis concomitantly with a parallel down-regulation of alpha 2MR/LRP expression. An up-regulation of uPAR expression was also observed. General endocytosis via the clathrin-coated pit pathway was not affected by PMA treatment, as judged from measurements of transferrin endocytosis. In LLC-PK1 cells, expressing alpha 2MR/LRP but not uPAR under basal conditions, phorbol esters transiently increase endocytosis in parallel with a transient induction of uPAR expression, while there was virtually no change in alpha 2MR/LRP expression. Differential regulation of endocytosis therefore seems to be caused by differential regulation of the receptors, with either the alpha 2MR/LRP-level (in COS)-1 cells) or the uPAR-level (in LLC-PK1 cells) being rate-limiting.
Asunto(s)
Endocitosis/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/farmacología , Receptores de Superficie Celular/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Línea Celular , Línea Celular Transformada , Chlorocebus aethiops , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Riñón/citología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Receptores de Superficie Celular/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa , PorcinosRESUMEN
The present report describes a new general procedure by which linear and some structure-dependent epitopes may be mapped in a protein antigen using a nested set of protein fragments prepared from partial proteolysis products of a recombinant protein. Briefly, the antigen, fused to an affinity tag, is partially fragmented and affinity sorted under denaturing conditions to produce a nested set of polypeptides, consisting of N- (or C-)terminal fragments. Immunoblots of SDS-PAGE fractionated sets of fragments are therefore directly readable in terms of molecular mass--i.e., approximate sequence positions--that identify sequence segments harbouring an epitope and any additional structural elements, required to maintain epitope conformation. Blots of N- and C-terminal nested sets of polypeptide fragments representing the human receptor associated protein (RAP) were prepared and probed with mAb S4D5 (Moestrup and Gliemann, 1991). Fragments 1-177 and 94-323 were the shortest fragments detected by the antibody, suggesting the presence of an epitope within the 94-177 segment. Independent mapping based on recombinant fragments of the RAP homologue, rat Heymann nephritis antigen, confirmed that the epitope resides in the Pro115-Asp177 segment. The model study demonstrates the utility of nested sets of protein fragments as fast and inexpensive tools for epitope mapping.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/análisis , Glicoproteínas de Membrana/inmunología , Fragmentos de Péptidos/inmunología , Mapeo Peptídico/métodos , Receptores Inmunológicos/inmunología , Secuencia de Aminoácidos , Animales , Autoantígenos/inmunología , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Complejo Antigénico de Nefritis de Heymann , Humanos , Immunoblotting , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Datos de Secuencia Molecular , RatasRESUMEN
We here report that the M(r) 40,000 receptor associated protein (RAP), previously found to bind to alpha 2-macroglobulin receptor/low density lipoprotein receptor related protein (alpha 2MR/LRP) and glycoprotein 330 (gp330), binds to an M(r) 105,000 membrane protein from bovine mammary gland, human mamma tumors and mammary epithelial cell lines. We have purified this protein from bovine and human sources. N-terminal amino acid sequencing and immunoblotting analyses showed that the protein was identical or closely related to very low density lipoprotein receptor (VLDL-R). Experiments with the human mamma carcinoma cell line MCF-7 showed that this receptor was able to mediate an efficient endocytosis of RAP. These novel findings strongly suggest that RAP functions as a modulator of ligand binding to VLDL-R, similarly to alpha 2MR/LRP and gp330.