RESUMO
We work with policymakers in eight cities worldwide to identify technology pathways toward their near- and long-term carbon emissions reduction targets for existing buildings. Based on policymakers' interests, we define city-specific shallow and deep retrofitting packages along with onsite photovoltaic generation potential. Without further grid decarbonization measures, stock-wide implementation of these retrofits in the investigated neighborhoods reduces energy use and carbon emissions by up to 66% and 84%, respectively, helping Braga, Dublin, Florianopolis, Middlebury, and Singapore to meet their 2030 goals. With projected grid decarbonization, Florianopolis and Singapore will reach their 2050 goals. The remaining emissions stem from municipalities not planning to electrify heating and/or domestic hot water use. Different climates and construction practices lead to varying retrofit packages, suggesting that comparable technology pathway analyses should be conducted for municipalities worldwide. Twenty months after the project ended, seven cities have implemented policy measures or expanded the analysis across their building stock.
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Membrane proteins play an essential role in all living organisms. Although there have been numerous efforts in the past to elucidate the structure and function of eukaryotic primary active transporters, knowledge about the majority of these membrane proteins is still minimal. This is often due to their low availability and complex handling. In this study, we homologously expressed three ATP-dependent transport proteins, STE6-2p, NEO1-p, and YPK9-p, in Pichia pastoris and subsequently optimized the solubilization and purification processes. Sequential use of different mild detergents and utilization of hydrophilic matrices in the purification procedure allowed us to obtain all three transporters monodisperse and in high purity, enabling initial structural analysis by cryo-electron microscopy. Using the respective substrates, we determined the specific activity of all target proteins using an ATPase assay. This study opens the door to further functional and structural studies of this pharmacologically important class of membrane proteins.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Pichia , Microscopia Crioeletrônica , Pichia/genética , Pichia/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificaçãoRESUMO
Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast Pichia pastoris, a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated the Pichia ABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter from P. pastoris in the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Esteróis , Humanos , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenilil Imidodifosfato/metabolismo , Esteróis/metabolismo , Filogenia , Trifosfato de Adenosina/metabolismo , Saccharomyces cerevisiae/metabolismo , Verapamil/farmacologia , Verapamil/metabolismo , Triazóis/metabolismo , Rodaminas/metabolismoRESUMO
Accurate occupancy is crucial for planning for sustainable buildings. Using massive, passively-collected mobile phone data, we introduce a novel framework to estimate building occupancy at unprecedented scale. We show that, at urban-scale, occupancy differs widely from current estimates based on building types. For commercial buildings, we find typical occupancy rates are 5 times lower than current assumptions imply, while for residential buildings occupancy rates vary widely by neighborhood. Our mobile phone based occupancy estimates are integrated with a state-of-the-art urban building energy model to understand their impact on energy use predictions. Depending on the assumed relationship between occupancy and internal building loads, we find energy consumption which differs by +1% to -15% for residential buildings and by -4% to -21% for commercial buildings, compared to standard methods. This highlights a need for new occupancy-to-load models which can be applied at urban-scale to the diverse set of city building types.
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Coagulation factor XIII (FXIII) is a plasma-circulating heterotetrameric pro-transglutaminase complex that is composed of two catalytic FXIII-A and two protective/regulatory FXIII-B subunits. FXIII acts by forming covalent cross-links within a preformed fibrin clots to prevent its premature fibrinolysis. The FXIII-A subunit is known to have pleiotropic roles outside coagulation, but the FXIII-B subunit is a relatively unexplored entity, both structurally as well as functionally. Its discovered roles so far are limited to that of the carrier/regulatory protein of its partner FXIII-A subunit. In the present study, we have explored the co-presence of protein excipients in commercial FXIII plasma concentrate FibrogamminP by combination of protein purification and mass spectrometry-based verification. Complement factor H was one of the co-excipients observed in this analysis. This was followed by performing pull down assays from plasma in order to detect the putative novel interacting partners for the FXIII-B subunit. Complement system proteins, like complement C3 and complement C1q, were amongst the proteins that were pulled down. The only protein that was observed in both experimental set ups was alpha-2-macroglobulin, which might therefore be a putative interacting partner of the FXIII/FXIII-B subunit. Future functional investigations will be needed to understand the physiological significance of this association.
Assuntos
Transtornos da Coagulação Sanguínea/etiologia , Transtornos da Coagulação Sanguínea/metabolismo , Proteínas de Transporte/metabolismo , Fator XIII/metabolismo , Mapeamento de Interação de Proteínas , Coagulação Sanguínea , Transtornos da Coagulação Sanguínea/sangue , Fator H do Complemento/metabolismo , Fibrinogênio/metabolismo , Humanos , Espectrometria de Massas , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Ligação ProteicaRESUMO
G-protein-coupled receptors (GPCRs) are the most important signal transducers in higher eukaryotes. Despite considerable progress, the molecular basis of subtype-specific ligand selectivity, especially for peptide receptors, remains unknown. Here, by integrating DNP-enhanced solid-state NMR spectroscopy with advanced molecular modeling and docking, the mechanism of the subtype selectivity of human bradykinin receptors for their peptide agonists has been resolved. The conserved middle segments of the bound peptides show distinct conformations that result in different presentations of their N and C termini toward their receptors. Analysis of the peptide-receptor interfaces reveals that the charged N-terminal residues of the peptides are mainly selected through electrostatic interactions, whereas the C-terminal segments are recognized via both conformations and interactions. The detailed molecular picture obtained by this approach opens a new gateway for exploring the complex conformational and chemical space of peptides and peptide analogs for designing GPCR subtype-selective biochemical tools and drugs.
Assuntos
Cininas/química , Receptor B1 da Bradicinina/química , Receptor B2 da Bradicinina/química , Receptores Acoplados a Proteínas G/química , Eletricidade Estática , Animais , Células HEK293 , Humanos , Insetos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Acoplamento Molecular , Mutação , Peptídeos/química , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Células Sf9 , Transdução de SinaisRESUMO
INTRODUCTION: The incidence rate of lung adenocarcinoma (LUAD), the predominant histological subtype of lung cancer, is elevated in Asians, particularly in female nonsmokers. The mutation patterns in LUAD in Asians might be distinct from those in LUAD in whites. METHODS: We profiled 271 resected LUAD tumors (mainly stage I) to characterize the genomic landscape of LUAD in Asians with a focus on female nonsmokers. RESULTS: Mutations in EGFR, KRAS, erb-b2 receptor tyrosine kinase 2 gene (ERBB2), and BRAF; gene fusions involving anaplastic lymphoma receptor tyrosine kinase gene (ALK), ROS1, and ret proto-oncogene (RET); and Met Proto-Oncogene Tyrosine Kinase (MET) exon 14 skipping were the major drivers in LUAD in Asians, exhibiting mutually exclusive and differing prevalence from those reported in studies of LUAD in non-Asians. In addition, we identified a novel mutational signature of XNX (the mutated base N in the middle flanked by two identical bases at the 5' and 3' positions) that was overrepresented in LUAD tumors in nonsmokers and negatively correlated with the overall mutational frequency. CONCLUSIONS: In this cohort, approximately 85% of individuals have known driver mutations (EGFR 59.4%, KRAS 7.4%, ALK 7.4%, ERBB2 2.6%, ROS1 2.2%, RET 2.2%, MET 1.8%, BRAF 1.1%, and NRAS 0.4%). Seventy percent of smokers and 90% of nonsmokers had defined oncogenic drivers matching the U.S. Food and Drug Administration-approved targeted therapies.
Assuntos
Adenocarcinoma/genética , Povo Asiático/genética , Carcinogênese/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Proto-Oncogene Mas , Adulto JovemRESUMO
The activation and regulation of coagulation Factor XIII (FXIII) protein has been the subject of active research for the past three decades. Although discrete evidence exists on various aspects of FXIII activation and regulation a combinatorial structure/functional view in this regard is lacking. In this study, we present results of a structure/function study of the functional chain of events for FXIII. Our study shows how subtle chronological submolecular changes within calcium binding sites can bring about the detailed transformation of the zymogenic FXIII to its activated form especially in the context of FXIIIA and FXIIIB subunit interactions. We demonstrate what aspects of FXIII are important for the stabilization (first calcium binding site) of its zymogenic form and the possible modes of deactivation (thrombin mediated secondary cleavage) of the activated form. Our study for the first time provides a structural outlook of the FXIIIA2B2 heterotetramer assembly, its association and dissociation. The FXIIIB subunits regulatory role in the overall process has also been elaborated upon. In summary, this study provides detailed structural insight into the mechanisms of FXIII activation and regulation that can be used as a template for the development of future highly specific therapeutic inhibitors targeting FXIII in pathological conditions like thrombosis.
Assuntos
Fator XIII/metabolismo , Sítios de Ligação/fisiologia , Cálcio/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Subunidades Proteicas/metabolismo , Trombina/metabolismo , Trombose/metabolismoRESUMO
In humans and other vertebrate animals, vitamin K 2,3-epoxide reductase (VKOR) family enzymes are the gatekeepers between nutritionally acquired K vitamins and the vitamin K cycle responsible for posttranslational modifications that confer biological activity upon vitamin K-dependent proteins with crucial roles in hemostasis, bone development and homeostasis, hormonal carbohydrate regulation and fertility. We report a phylogenetic analysis of the VKOR family that identifies five major clades. Combined phylogenetic and site-specific conservation analyses point to clade-specific similarities and differences in structure and function. We discovered a single-site determinant uniquely identifying VKOR homologs belonging to human pathogenic, obligate intracellular prokaryotes and protists. Building on previous work by Sevier et al. (Protein Science 14:1630), we analyzed structural data from both VKOR and prokaryotic disulfide bond formation protein B (DsbB) families and hypothesize an ancient evolutionary relationship between the two families where one family arose from the other through a gene duplication/deletion event. This has resulted in circular permutation of primary sequence threading through the four-helical bundle protein folds of both families. This is the first report of circular permutation relating distant a-helical membrane protein sequences and folds. In conclusion, we suggest a chronology for the evolution of the five extant VKOR clades.
Assuntos
Evolução Biológica , Dissulfetos , Filogenia , Receptores Fc/genética , Vitamina K Epóxido Redutases/genética , Vitamina K/metabolismo , Sequência de Aminoácidos , Animais , Archaea , Bactérias , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Vitamina K Epóxido Redutases/químicaRESUMO
Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K1 2,3-epoxide (K1>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis-Menten constants of 1.20 µM for K1>O and 260 µM for the active neutral form of THPP. The Km value for K1>O is in agreement with the value that we previously obtained using DTT (1.24 µM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K1 and its previously reported base-catalyzed conversion to K1, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.
Assuntos
Ditiotreitol/metabolismo , Fosfinas/metabolismo , Vitamina K Epóxido Redutases/metabolismo , Biocatálise , Soluções Tampão , Ácidos Cólicos/metabolismo , Ensaios Enzimáticos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Pichia/metabolismo , Substâncias Redutoras/metabolismo , Soluções , Especificidade por SubstratoRESUMO
BACKGROUND: Warfarin directly inhibits vitamin K 2,3-epoxide reductase (VKOR) enzymes. Since the early 1970s, warfarin inhibition of vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), an essential enzyme for proper function of blood coagulation in higher vertebrates, has been studied using an in vitro dithiothreitol (DTT) driven enzymatic assay. However, various studies based on this assay have reported warfarin dose-response data, usually summarized as half-maximal inhibitory concentration (IC50), that vary over orders of magnitude and reflect the broad range of conditions used to obtain VKOR assay data. METHODS: We standardized the implementation of the DTT-driven VKOR activity assay to measure enzymatic Michaelis constants (Km) and warfarin IC50 for human VKORC1. A data transformation is defined, based on the previously confirmed bi bi ping-pong mechanism for VKORC1, that relates assay condition-dependent IC50 to condition-independent Ki. RESULTS: Determination of the warfarin Ki specifically depends on measuring both substrate concentrations, both Michaelis constants for the VKORC1 enzyme, and pH in the assay. CONCLUSION: The Ki is not equal to the IC50 value directly measured using the DTT-driven VKOR assay. GENERAL SIGNIFICANCE: In contrast to warfarin IC50 values determined in previous studies, warfarin inhibition expressed as Ki can now be compared between studies, even when the specific DTT-driven VKOR assay conditions differ. This implies that warfarin inhibition reported for wild-type and variant VKORC1 enzymes from previous reports should be reassessed and new determinations of Ki are required to accurately report and compare in vitro warfarin inhibition results.
Assuntos
Ditiotreitol/farmacologia , Oxigenases de Função Mista/antagonistas & inibidores , Varfarina/farmacologia , Humanos , Cinética , Vitamina K Epóxido RedutasesRESUMO
Recent success in obtaining high-resolution structural data for the first several G protein-coupled receptors (GPCRs) has highlighted the feasibility of structural membrane proteomics approaches for obtaining molecular models of additional GPCRs from among the nearly 800 encoded by the human genome. Yet, production of functional receptors, in general, has proven to be difficult, typically requiring considerable time and cost investments. Here we describe screening, optimization, and scale-up methods we successfully used to produce milligram amounts of functional GPCRs in Pichia pastoris. When we surveyed a large number of receptors recombinantly produced in Pichia, 85% exhibited specific ligand binding, strongly suggesting that this expression system is excellent for producing functional GPCRs. Of the latter group, 20 were optimized according to our protocol. Of these, we produced 10 as milligram amounts of functional receptors using large-scale shaker culture. Cost and time expenditures were considerably lower using the Pichia system than for other successfully employed cell culture systems.
Assuntos
Pichia/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Pichia/genética , Receptores Acoplados a Proteínas G/genética , Transformação GenéticaRESUMO
Human Neuromedin U receptor 1 (hNmU-R1) is a member of G protein-coupled receptor family. For structural determination of hNmU-R1, the production of hNmU-R1 in milligram amounts is a prerequisite. Here we reported two different eukaryotic expression systems, namely, Semliki Forest virus (SFV)/BHK-21 and baculovirus/Spodoptera frugiperda (Sf9) cell systems for overproduction of this receptor. In the SFV-based expression system, hNmU-R1 was produced at a level of 5 pmol receptor/mg membrane protein and the yield could be further increased to 22 pmol receptor/mg membrane protein by supplementation with 2% dimethyl sulfoxide (DMSO). Around 8 pmol receptor/mg membrane protein could be achieved in baculovirus-infected Sf9 cells. The recombinant hNmU-R1 from SFV- and baculovirus-based systems was functional, with a Kd value of [125I] NmU-23 (rat) similar to that from transiently transfected COS-7 cells, where hNmU-R1 was first identified. With the aid of 1% n-dodecyl-beta-D-maltoside (LM)/0.25% cholesteryl hemisuccinate (CHS), the yield of functional hNmU-R1 could reach 80%. The recombinant receptor from Sf9 cells was purified to homogeneity. The specific binding of the purified receptor to [125I] NmU-23 (rat) indicated that the receptor is bioactive. This is the first report of successful solubilization and purification of hNmU-R1, and will enable functional and structural studies of the hNmU-R1.
Assuntos
Receptores de Neurotransmissores/isolamento & purificação , Receptores de Neurotransmissores/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Linhagem Celular , Humanos , Ratos , Receptores de Neurotransmissores/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/metabolismo , Spodoptera/genética , Spodoptera/metabolismoRESUMO
We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptor CB1 de Canabinoide/química , Receptor CB1 de Canabinoide/metabolismo , Spodoptera/citologia , Spodoptera/metabolismo , Animais , Baculoviridae/genética , Membrana Celular/química , Células Cultivadas , Dimerização , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Vetores Genéticos , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Microscopia Confocal , Peso Molecular , Piperidinas/farmacologia , Testes de Precipitina , Ligação Proteica , Pirazóis/farmacologia , Ensaio Radioligante , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Rimonabanto , Proteínas Virais de Fusão/metabolismoRESUMO
The human bradykinin subtype 2 receptor (B(2)R), a member of class A GPCRs, was heterologously expressed in the methylotrophic yeast Pichia pastoris. The recombinant receptor was produced as a fusion protein with affinity tags and it was expressed at a level of 3.5 pmol/mg of total membrane protein. [(3)H]Bradykinin binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (K(d)=0.87+/-0.1 nM), similar to the native receptor. Enzymatic deglycosylation revealed that the recombinant B(2)R was produced in a glycosylated form. Immunogold staining of the Pichia cells expressing B(2)R suggested that the recombinant receptor was localized intracellularly and it was not present in the plasma membrane. The data presented here should facilitate isolation of the recombinant receptor for structural studies.
Assuntos
Pichia/genética , Receptor B2 da Bradicinina/biossíntese , Proteínas Recombinantes/biossíntese , Clonagem Molecular , Glicosilação , Humanos , Imuno-Histoquímica , Metano/metabolismo , Pichia/química , Receptor B2 da Bradicinina/química , Receptor B2 da Bradicinina/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/químicaRESUMO
Neuromedin U (a neuropeptide) plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow and pain. The physiological actions of NmU are mediated via two recently identified G protein-coupled receptors namely the neuromedin U type 1 receptor (NmU(1)R) and the neuromedin U type 2 receptor (NmU(2)R). Despite their crucial roles in cell physiology, structural information on these receptors is limited, mainly due to their low expression levels in native tissues. Here, we report the overexpression of the human NmU(2)R in the methylotrophic yeast Pichia pastoris and baby hamster kidney (BHK) cells using the Semliki Forest virus (SFV) system. The recombinant receptor was expressed as a fusion protein with three different affinity tags namely, the Flag tag, the histidine 10 tag and the biotinylation domain of Propionobacterium shermanii. Expression level of the recombinant receptor was 6-9pmol/mg under optimized conditions, which is significantly higher than the expression level in the native tissues. The recombinant receptor binds to its endogenous ligand neuromedin U with high affinity (Kd=0.8-1.0nM) and the binding constant for the recombinant receptor is similar to that of the wild type NmU(2)R. Enzymatic deglycosylation suggested that the recombinant NmU(2)R was glycosylated in P. pastoris, but not in BHK cells. Confocal laser scanning microscopy and immunogold labelling experiment revealed that the recombinant receptor was predominantly localized in the intracellular membranes. To our knowledge, this is the first report of heterologous overexpression of an affinity tagged recombinant NmU(2)R and it should facilitate further characterization of this receptor.
Assuntos
Proteínas de Membrana/metabolismo , Pichia/genética , Receptores de Neurotransmissores/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , Dimetil Sulfóxido/farmacologia , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/genética , Glicosilação , Radioisótopos do Iodo , Cinética , Proteínas de Membrana/genética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Neuropeptídeos/metabolismo , Pichia/metabolismo , Pichia/ultraestrutura , Ligação Proteica , Receptores de Neurotransmissores/genética , Proteínas Recombinantes/genéticaRESUMO
Production of recombinant receptors has been one of the major bottlenecks in structural biology on G protein-coupled receptors (GPCRs). The MePNet (Membrane Protein Network) was established to overexpress a large number of GPCRs in three major expression systems, based on Escherichia coli, Pichia pastoris and Semliki Forest virus (SFV) vectors. Evaluation by immunodetection demonstrated that 50% of a total of 103 GPCRs were expressed in bacterial inclusion bodies, 94% in yeast cell membranes and 95% in SFV-infected mammalian cells. The expression levels varied from low to high and the various GPCR families and subtypes were analyzed for their expressability in each expression system. More than 60% of the GPCRs were expressed at milligram levels or higher in one or several systems, compatible to structural biology applications. Functional activity was determined by binding assays in yeast and mammalian cells and the correlation between immunodetection and binding activity was analyzed.
Assuntos
Escherichia coli , Expressão Gênica , Genômica , Proteínas de Membrana , Pichia , Receptores Acoplados a Proteínas G , Vírus da Floresta de Semliki , Animais , Western Blotting , Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Genes Fúngicos , Genes Virais , Vetores Genéticos , Humanos , Immunoblotting , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Pichia/genética , Ligação Proteica , Ensaio Radioligante , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Vírus da Floresta de Semliki/genéticaRESUMO
Angiotensin II type 1a receptor (AT1aR) is a member of GPCR superfamily and it plays crucial roles in the regulation of blood pressure, hormone secretion and renal functions. Here, we report functional overexpression and characterization of the human AT1aR in insect cells using the baculovirus system and in mammalian cells using the Semliki Forest virus system. The recombinant receptor was expressed at a level of 29-32 pmol/mg and it binds to angiontensin II with high affinity (Kd=0.98-1.1 nM). Angiotensin II stimulated accumulation of inositol phosphate and phosphorylation of MAP kinase was also observed, which indicated that the recombinant AT1aR could couple to endogenous Galphaq protein. Confocal laser scanning microscopy revealed that the recombinant receptor was predominantly localized in the plasma membrane and agonist induced internalization of the recombinant AT1aR was also observed. The recombinant AT1aR was expressed in glycosylated form and in vivo inhibition of glycosylation suppressed its surface expression.
Assuntos
Técnicas Genéticas , Receptor Tipo 1 de Angiotensina/genética , Animais , Membrana Celular/metabolismo , Cricetinae , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Glicosilação , Humanos , Insetos , Microscopia Confocal , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Vírus da Floresta de Semliki/genéticaRESUMO
One of the major objectives of the Membrane Protein Network program (MePNet, www.mepnet.org) is to express one hundred G-protein-coupled receptors (GPCRs) in the yeast Pichia pastoris. We have developed an antibody-based assay in order to select for the best behaving clones at each step of the receptor preparation, from expression to solubilization. This assay allowed us to quantify the expression of Flag-tagged GPCRs present in various sample types, from crude P. pastoris extracts to native membrane preparations and detergent solubilised fractions. It combines the specificity of ELISA, the sensitivity of enhanced chemiluminescence detection and the speed of high-throughput screening, and can detect as low as 0.001% (w/w) flag-tagged recombinant receptor present in a crude extract. The method was applied to sort recombinant GPCR clones, to rank receptor expression levels and to screen for detergent solubilization efficiency using the human beta2-adrenergic receptor expressed in P. pastoris as a benchmarking standard.