RESUMO
Tau hyperphosphorylation has been linked directly to the formation of toxic neurofibrillary tangles (NFTs) in tauopathies, however, prior to NFT formation, the sequence of pathological events involving tau phosphorylation remains unclear. Here, the effect of glycogen synthase kinase 3ß (GSK3ß) on tau pathology was examined independently for each step of transcellular propagation; namely, tau intracellular aggregation, release, cellular uptake and seeding activity. We find that overexpression of GSK3ß-induced phosphorylated 0N4R tau led to a higher level of tau oligomerization in SH-SY5Y neuroblastoma cells than wild type 0N4R, as determined by several orthogonal assays. Interestingly, the presence of GSK3ß also enhanced tau release. Further, we demonstrated that cells endocytosed more monomeric tau protein when pre-phosphorylated by GSK3ß. Using an extracellular vesicle (EVs)-assisted tau neuronal delivery system, we show that exosomal GSK3ß-phosphorylated tau, when added to differentiated SH-SY5Y cells, induced more efficient tau transfer, showing much higher total tau levels and increased tau aggregate formation as compared to wild type exosomal tau. The role of a primary tau phosphorylation site targeted by microtubule-affinity regulating kinases (MARKs), Ser262, was tested by pseudo-phosphorylation using site-directed mutagenesis to aspartate (S262D). S262D tau overexpression significantly enhanced tau release and intracellular tau accumulation, which were concurrent with the increase of pathological states of tau, as determined by immunodetection. Importantly, phosphorylation-induced tau accumulation was augmented by co-transfecting S262D tau with GSK3ß, suggesting a possible interplay between Ser262 phosphorylation and GSK3ß activity in tau pathology. Lastly, we found that pre-treatment of cells with amyloid-ß (Aß) further tau phosphorylation and accumulation when Ser262 pre-phosphorylation was present, suggesting that S262 may be a primary mediator of Aß-induced tau toxicity. These findings provide a potential therapeutic target for treating tau-related disorders by targeting specific phospho-tau isoforms and further elucidate the GSK3ß-mediated pathological seeding mechanisms.
Assuntos
Neuroblastoma , Proteínas tau , Peptídeos beta-Amiloides/metabolismo , Ácido Aspártico/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Fosforilação , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
BACKGROUND: Membrane cholesterol dysregulation has been shown to alter the activity of the adenosine A2A receptor (A2AR), a G protein-coupled receptor, thereby implicating cholesterol levels in diseases such as Alzheimer's and Parkinson's. A limited number of A2AR crystal structures show the receptor interacting with cholesterol, as such molecular simulations are often used to predict cholesterol interaction sites. METHODS: Here, we use experimental methods to determine whether a specific interaction between amino acid side chains in the cholesterol consensus motif (CCM) of full length, wild-type human A2AR, and cholesterol modulates activity of the receptor by testing the effects of mutational changes on functional consequences, including ligand binding, G protein coupling, and downstream activation of cyclic AMP. RESULTS AND CONCLUSIONS: Our data, taken with previously published studies, support a model of receptor state-dependent binding between cholesterol and the CCM, whereby cholesterol facilitates both G protein coupling and downstream signaling of A2AR.
Assuntos
Adenosina , Receptor A2A de Adenosina , Adenosina/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Proteínas de Transporte , Colesterol/metabolismo , AMP Cíclico/metabolismo , Humanos , Receptor A2A de Adenosina/metabolismoRESUMO
The adenosine A2A receptor (A2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilization in DDM, DDM/CHAPS, or DHPC micelles, although A2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. The studies presented in this paper also underline the importance of the protein's purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.
Assuntos
Ésteres do Colesterol/química , Ácidos Cólicos/química , Éteres Fosfolipídicos/química , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/isolamento & purificação , Proteínas Recombinantes/química , Humanos , Estrutura Secundária de ProteínaRESUMO
γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) â« 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) â¼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.
Assuntos
Mutação Puntual , gama-Cristalinas/química , gama-Cristalinas/genética , Sequência de Aminoácidos , Dicroísmo Circular , Desenho Assistido por Computador , Humanos , Cinética , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Termodinâmica , gama-Cristalinas/metabolismoRESUMO
Tauopathies represent a group of neurodegenerative diseases including Alzheimer's disease (AD) that are characterized by the deposition of filamentous tau aggregates in the brain. The pathogenesis of tauopathies starts from the formation of toxic 'tau seeds' from hyperphosphorylated tau monomers. The presence of specific phosphorylation sites and heat shock protein 90 facilitates soluble tau protein aggregation. Transcellular propagation of pathogenic tau into synaptically connected neuronal cells or adjacent glial cells via receptor-mediated endocytosis facilitate disease spread through the brain. While neuroprotective effects of glial cells-including phagocytotic microglial and astroglial phenotypes-have been observed at the early stage of neurodegeneration, dysfunctional neuronal-glial cellular communication results in a series of further pathological consequences as the disease progresses, including abnormal axonal transport, synaptic degeneration, and neuronal loss, accompanied by a pro-inflammatory microenvironment. Additionally, the discovery of microtubule-associated protein tau (MAPT) gene mutations and the strongest genetic risk factor of tauopathies-an increase in the presence of the ε2 allele of apolipoprotein E (ApoE)-provide important clues to understanding tau pathology progression. In this review, we describe the crucial signaling pathways and diverse cellular contributors to the progression of tauopathies. A systematic understanding of disease pathogenesis provides novel insights into therapeutic targets within altered signaling pathways and is of great significance for discovering effective treatments for tauopathies.
RESUMO
TSP (P22 tailspike protein) is a well-established model system for studying the folding and assembly of oligomeric proteins, and previous studies have documented both in vivo and in vitro folding intermediates using this protein. Especially important is the C-terminus of TSP, which plays a critical role in the assembly and maturation of the protrimer intermediate to its final trimeric form. In the present study, we show that by grafting the C-terminus of TSP on to the monomeric MBP (maltose-binding protein), the resulting chimaera (MBP-537) is a trimeric protein. Moreover, Western blot studies (using an anti-TSP antibody) indicate that the TSP C-terminus in the MBP-537 chimaera has the same conformation as the native TSP. The oligomerization of the MBP-537 chimaera appears to involve hydrophobic interactions and a refolding sequence, both of which are analogous to the native TSP. These results underscore the importance of the TSP C-terminus in the assembly of the mature trimer and demonstrate its potential utility as a model to study the folding and assembly of the TSP C-terminus in isolation.
Assuntos
Bacteriófago P22/química , Multimerização Proteica , Proteínas da Cauda Viral/química , Anticorpos/imunologia , Western Blotting , Proteínas de Transporte/metabolismo , Centrifugação , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases , Interações Hidrofóbicas e Hidrofílicas , Proteínas Ligantes de Maltose , Proteínas Mutantes/metabolismo , Mutação/genética , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas da Cauda Viral/imunologia , Proteínas da Cauda Viral/metabolismoRESUMO
Heterologous protein expression can easily overwhelm a cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. In the current model of the UPR, the chaperone BiP modulates the activation of the UPR due to its interactions with the signaling protein Ire1p and newly synthesized proteins. In this research, 4-4-20 scFv variants were generated by rational design to alter BiP binding to newly synthesized scFv proteins or via directed evolution aimed at improved secretion. Interestingly, the predicted BiP binding ability did not correlate significantly with the UPR. However, pulse-chase analysis of scFv fate revealed that mutants with a decreased ER residence time were more highly secreted, indicating that improved protein folding was more likely the cause for improved secretion. In fact, decreased secretion correlated with increased binding by BiP, as determined by co-immune precipitation studies. This suggests that the algorithm is not useful for in vivo prediction of variants, and that in vivo screens are more effective for finding variants with improved properties.
Assuntos
Anticorpos/metabolismo , Proteínas Recombinantes/metabolismo , Saccharomyces/metabolismo , Anticorpos/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Imunoprecipitação/métodos , Modelos Moleculares , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes/genéticaRESUMO
Cholesterol has been shown to modulate the activity of multiple G Protein-coupled receptors (GPCRs), yet whether cholesterol acts through specific interactions, indirectly via modifications to the membrane, or via both mechanisms is not well understood. High-resolution crystal structures of GPCRs have identified bound cholesterols; based on a ß2-adrenergic receptor (ß2AR) structure bound to cholesterol and the presence of conserved amino acids in class A receptors, the cholesterol consensus motif (CCM) was identified. Here in mammalian cells expressing the adenosine A2A receptor (A2AR), ligand dependent production of cAMP is reduced following membrane cholesterol depletion with methyl-beta-cyclodextrin (MßCD), indicating that A2AR signaling is dependent on cholesterol. In contrast, ligand binding is not dependent on cholesterol depletion. All-atom molecular simulations suggest that cholesterol interacts specifically with the CCM when the receptor is in an active state, but not when in an inactive state. Taken together, the data support a model of receptor state-dependent binding between cholesterol and the CCM, which could facilitate both G-protein coupling and downstream signaling of A2AR.
Assuntos
Resina de Colestiramina , Simulação de Dinâmica Molecular , Receptor A2A de Adenosina , Transdução de Sinais , Resina de Colestiramina/química , Resina de Colestiramina/metabolismo , Células HEK293 , Humanos , Receptor A2A de Adenosina/química , Receptor A2A de Adenosina/metabolismo , beta-Ciclodextrinas/químicaRESUMO
A top-down approach to mechanistic modeling of biological systems is presented and exemplified with the development of a hypothesis-driven mathematical model for single-chain antibody fragment (scFv) folding in Saccharomyces cerevisiae by mediators BiP and PDI. In this approach, model development starts with construction of the most basic mathematical model--typically consisting of predetermined or newly-elucidated biological behavior motifs--capable of reproducing desired biological behaviors. From this point, mechanistic detail is added incrementally and systematically, and the effects of each addition are evaluated. This approach follows the typical progression of experimental data availability in that higher-order, lumped measurements are often more prevalent initially than specific, mechanistic ones. It also necessarily provides the modeler with insight into the structural requirements and performance capabilities of the resulting detailed mechanistic model, which facilitates further analysis. The top-down approach to mechanistic modeling identified three such requirements and a branched dependency-degradation competition motif critical for the scFv folding model to reproduce experimentally observed scFv folding dependencies on BiP and PDI and increased production when both species are overexpressed and promoted straightforward prediction of parameter dependencies. It also prescribed modification of the guiding hypothesis to capture BiP and PDI synergy.
Assuntos
Anticorpos/química , Anticorpos/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/metabolismo , Modelos Biológicos , Dobramento de Proteína , Biologia Computacional , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Cinética , Chaperonas Moleculares/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismoRESUMO
Heterotrimeric G proteins relay signals from G protein-coupled receptors (GPCRs) to the interior of the cell. The signaling cascades induced by G protein activation control a wide range of cellular processes. The alpha subunit is believed to determine which G protein couples to each GPCR, and is the primary determinant of the type of signal transmitted. Several members of the G(alpha) family have been expressed in active form in Escherichia coli. However, production levels of these proteins are limited: in most cases only approximately 10% of total G(alpha) protein expressed is active; the rest accumulates in inclusion bodies. Although G(ialpha) has been readily expressed in soluble form (to 10 mg/L), other alpha subunits are minimally soluble, and many are exclusively expressed to inclusion bodies. Previous efforts to solubilize and refold G(alpha) from inclusion bodies have not been successful. Here we did a thorough study of the characteristics of G(alpha) subunits (human G(ialpha(1)), human G(salpha(short)), human G(11alpha) and human G(talpha(cone))), solubilized and purified from inclusion bodies. We find that we can obtain soluble protein both by on-column and rapid-dilution techniques. Comparison to native, soluble G(ialpha) expressed from E. coli showed that although the refolded G(alpha) subunits were soluble and retained partial alpha-helicity characteristic of the native, folded G(alpha) subunit, they did not bind GDP or GTP as effectively as native protein. We conclude that the refolded G(ialpha) protein has a native-like secondary structure, but is predominantly in a molten globular state.
Assuntos
Escherichia coli/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Corpos de Inclusão/metabolismo , Dicroísmo Circular , Subunidades alfa de Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Dobramento de Proteína , Estrutura Terciária de Proteína , Espectrofotometria UltravioletaRESUMO
Although manipulation of the endoplasmic reticulum (ER) folding environment in the yeast Saccharomyces cerevisiae has been shown to increase the secretory productivity of recombinant proteins, the cellular interactions and processes of native enzymes and chaperones such as protein disulfide isomerase (PDI) are still unclear. Previously, we reported that overexpression of the ER chaperone PDI enabled up to a 3-fold increase in secretion levels of the Pyrococcus furiosus beta-glucosidase in the yeast S. cerevisiae. This result was surprising since beta-glucosidase contains only one cysteine per monomer and no disulfide bonds. Two possible mechanisms were proposed: PDI either forms a transient disulfide bond with the lone cysteine residue of the nascent beta-glucosidase during the folding and assembly process or acts as a chaperone to aid in proper folding. To discern between the two mechanisms, the single cysteine residue was mutated to serine, and the secretion of the two protein variants was determined. The serine mutant still showed increased secretion in vivo when PDI levels were elevated. When the folding bottleneck is removed by increasing expression temperatures to 37 degrees C rather than 30 degrees C, PDI no longer has an improvement on secretion. These results suggest that, unexpectedly, PDI acts in a chaperone-like capacity or possibly cooperates with the cell's folding or degradation mechanisms regardless of whether the protein is redox-active.
Assuntos
Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Engenharia de Proteínas/métodos , Pyrococcus furiosus/fisiologia , Saccharomyces cerevisiae/fisiologia , beta-Glucosidase/genética , beta-Glucosidase/metabolismo , Melhoramento Genético/métodos , Oxirredução , Proteínas Recombinantes/metabolismoRESUMO
G protein-coupled receptors (GPCRs) are an important, medically relevant class of integral membrane proteins. Laboratories throughout all disciplines of science devote time and energy into developing practical methods for the discovery, isolation, and characterization of these proteins. Since the crystal structure of rhodopsin was solved 6 years ago, the race to determine high-resolution structures of more GPCRs has gained momentum. Since certain GPCRs are currently produced at sufficient levels for X-ray crystallography trials, it is speculated that heterologous expression of GPCRs may no longer be a bottleneck in obtaining crystal structures. This Review focuses on the current approaches in heterologous expression of GPCRs and explores the problems associated with obtaining crystal structures from GPCRs expressed in different systems. Although milligram amounts of certain GPCRs are attainable, the majority of GPCRs are still either produced at very low levels or not at all. Developing reliable expression techniques for GPCRs is still a major priority for the structural characterization of GPCRs.
Assuntos
Cristalografia por Raios X/métodos , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Animais , Escherichia coli/genética , Expressão Gênica , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Leveduras/genéticaRESUMO
Recombinant proteins offer many therapeutic advantages unavailable in traditional small molecule drugs, but the need for cellular versus chemical synthesis complicates production. Avenues for producing therapeutic biologics are continuously expanding, and developments in biochemistry, cell biology, and bioengineering fuel new discoveries that promise safer, more efficient, and cheaper drugs for consumers. Numerous approaches to express recombinant proteins exist, but Escherichia coli, Saccharomyces cerevisiae, and mammalian systems (e.g. Chinese hamster ovary cells, CHO) are the most widely utilized. Improvements to production in these hosts have focused on novel expression cassettes, cell line modifications, engineering secretion pathways, and media design. Here, we describe recent developments for improving protein production in E. coli, S. cerevisiae, and CHO systems and compare recent advancements to previous knowledge in the field. With the expanding importance and prevalence of protein therapeutics, these improvements will serve as the framework for future discoveries.
Assuntos
Engenharia Celular/métodos , Escherichia coli/genética , Engenharia de Proteínas/métodos , Saccharomyces cerevisiae/genética , Animais , Apoptose/fisiologia , Linhagem Celular , Meios de Cultura/química , Meios de Cultura/farmacologia , Escherichia coli/metabolismo , Humanos , Mamíferos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Saccharomyces cerevisiae/metabolismoRESUMO
Ligand binding plays a fundamental role in stimulating the downstream signaling of membrane receptors. Here, ligand-binding kinetics of the full-length human adenosine A2A receptor (A2AR) reconstituted in detergent micelles were measured using a fluorescently labeled ligand via fluorescence anisotropy. Importantly, to optimize the signal-to-noise ratio, these experiments were conducted in the ligand depletion regime. In the ligand depletion regime, the assumptions used to determine analytical solutions for one-site binding models for either one or two ligands in competition are no longer valid. We therefore implemented a numerical solution approach to analyze kinetic binding data as experimental conditions approach the ligand depletion regime. By comparing the results from the numerical and the analytical solutions, we highlight the ligand-receptor ratios at which the analytical solution begins to lose predictive accuracy. Using the numerical solution approach, we determined the kinetic rate constants of the fluorescent ligand, FITC-APEC, and those for three unlabeled ligands using competitive association experiments. The association and dissociation rate constants of the unlabeled ligands determined from the competitive association experiments were then independently validated using competitive dissociation data. Based on this study, a numerical solution is recommended to determine kinetic ligand-binding parameters for experiments conducted in the ligand-depletion regime.
Assuntos
Ligantes , Transporte Proteico/genética , Receptor A2A de Adenosina/química , Sítios de Ligação , Ligação Competitiva , Polarização de Fluorescência , Humanos , Cinética , Ligação Proteica , Receptor A2A de Adenosina/metabolismoRESUMO
Each chain of the native trimeric P22 tailspike protein has eight cysteines that are reduced and buried in its hydrophobic core. However, disulfide bonds have been observed in the folding pathway and they are believed to play a critical role in the registration of the three chains. Interestingly, in the presence of sodium dodecyl sulfate (SDS) only monomeric chains, rather than disulfide-linked oligomers, have been observed from a mixture of folding intermediates. Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.
Assuntos
Dissulfetos/química , Dodecilsulfato de Sódio/farmacologia , Proteínas da Cauda Viral/química , Dimerização , Eletroforese em Gel de Poliacrilamida , Glicosídeo Hidrolases , Dobramento de ProteínaRESUMO
The P22 tailspike protein folds by forming a folding competent monomer species that forms a dimeric, then a non-native trimeric (protrimer) species by addition of folding competent monomers. We have found three residues, R549, R563, and D572, which play a critical role in both the stability of the native tailspike protein and assembly and maturation of the protrimer. King and colleagues reported previously that substitution of R563 to glutamine inhibited protrimer formation. We now show that the R549Q and R563K variants significantly delay the protrimer-to-trimer transition both in vivo and in vitro. Previously, variants that destabilize intermediates have shown wild-type chemical stability. Interestingly, both the R549Q and R563K variants destabilize the tailspike trimer in guanidine denaturation studies, indicating that they represent a new class of tailspike folding variants. R549Q has a midpoint of unfolding at 3.2M guanidine, compared to 5.6M for the wild-type tailspike protein, while R563K has a midpoint of unfolding of 1.8 M. R549Q and R563K also denature over a broader pH range than the wild-type tailspike protein and both proteins have increased sensitivity to pH during refolding, suggesting that both residues are involved in ionic interactions. Our model is that R563 and D572 interact to stabilize the adjacent turn, aiding the assembly of the dimer and protrimer species. We believe that the interaction between R563 and D572 is also critical following assembly of the protrimer to properly orient D572 in order to form a salt bridge with R549 during protrimer maturation.
Assuntos
Proteínas da Cauda Viral/química , Substituição de Aminoácidos , Aminoácidos/química , Aminoácidos/genética , Glicosídeo Hidrolases , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas da Cauda Viral/genéticaRESUMO
The tailspike protein from the bacteriophage P22 is a well characterized model system for folding and assembly of multimeric proteins. Folding intermediates from both the in vivo and in vitro pathways have been identified, and both the initial folding steps and the protrimer-to-trimer transition have been well studied. In contrast, there has been little experimental evidence to describe the assembly of the protrimer. Previous results indicated that the C terminus plays a critical role in the overall stability of the P22 tailspike protein. Here, we present evidence that the C terminus is also the critical assembly point for trimer assembly. Three truncations of the full-length tailspike protein, TSPDeltaN, TSPDeltaC, and TSPDeltaNC, were generated and tested for their ability to form mixed trimer species. TSPDeltaN forms mixed trimers with full-length P22 tailspike, but TSPDeltaC and TSPDeltaNC are incapable of forming similar mixed trimer species. In addition, mutations in the hydrophobic core of the C terminus were unable to form trimer in vivo. Finally, the hydrophobic-binding dye ANS inhibits the formation of trimer by inhibiting progression through the folding pathway. Taken together, these results suggest that hydrophobic interactions between C-terminal regions of P22 tailspike monomers play a critical role in the assembly of the P22 tailspike trimer.
Assuntos
Proteínas da Cauda Viral/química , Dicroísmo Circular , Cristalografia por Raios X , Glicosídeo Hidrolases , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dobramento de Proteína , Renaturação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência/genética , Proteínas da Cauda Viral/genéticaRESUMO
Several temperature-sensitive folding (tsf) mutants of the tailspike protein from bacteriophage P22 have been found to fold with lower efficiency than the wild-type sequence, even at lowered temperatures. Previous refolding studies initiated from the unfolded monomer have indicated that the tsf mutations decrease the rate of structured monomer formation. We demonstrate that pressure treatment of the tailspike aggregates provides a useful tool to explore the effects of tsf mutants on the assembly pathway of the P22 tailspike trimer. The effects of pressure on two different tsf mutants, G244R and E196K, were explored. Pressure treatment of both G244R and E196K aggregates produced a folded trimer. E196K forms almost no native trimer in in vitro refolding experiments, yet it forms a trimer following pressure in a manner similar to the native tailspike protein. In contrast, trimer formation from pressure-treated G244R aggregates was not rapid, despite the presence of a G244R dimer after pressure treatment. The center-of-mass shifts of the fluorescence spectra under pressure are nearly identical for both tsf aggregates, indicating that pressure generates similar intermediates. Taken together, these results suggest that E196K has a primary defect in formation of the beta-helix during monomer collapse, while G244R is primarily an assembly defect.
Assuntos
Bacteriófago P22/química , Mutação/genética , Temperatura , Proteínas da Cauda Viral/química , Proteínas da Cauda Viral/genética , Bacteriófago P22/genética , Glicosídeo Hidrolases , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Pressão , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Renaturação ProteicaRESUMO
Recovering native protein from aggregates is a common obstacle in the production of recombinant proteins. Recent reports have shown that hydrostatic pressure is an attractive alternative to traditional denature-and-dilute techniques, both in terms of yield and process simplicity. To determine the effect of process variables, we subjected tailspike aggregates to a variety of pressure-treatment conditions. Maximum native tailspike yields were obtained with only short pressure incubations (<5 min) at 240 MPa. However, some tailspike aggregates were resistant to pressure, despite multiple cycles of pressure. Extending the postpressure incubation time to 4 days improved the yield of native protein from aggregates from 19.4 +/- 0.9 to 47.4 +/- 19.6 microg/mL (approximately 78% yield of native trimer from nonaggregate material). The nearly exclusive conversion of monomer to trimer over the time scale of days, when combined with previous kinetic data, allows for the identification of three postpressure kinetic phases: a rapid phase consisting of structured dimer conversion to trimer (30 min), an intermediate phase consisting of monomer conversion to aggregate (100 min), and a slow phase consisting of conversion of monomer to trimer (days). Optimizing the production of structured dimer can yield the highest level of folded protein. Typical refolding additives, such as glycerol, or low-temperature incubation did not improve yields.
Assuntos
Glicerol/química , Proteínas da Cauda Viral/química , Proteínas da Cauda Viral/isolamento & purificação , Misturas Complexas/química , Misturas Complexas/isolamento & purificação , Dimerização , Glicosídeo Hidrolases , Substâncias Macromoleculares , Pressão , Ligação Proteica , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Temperatura , Ureia/químicaRESUMO
Cells are inherently robust to environmental perturbations and have evolved to recover readily from short-term exposure to heat, pH changes, and nutrient deprivation during times of stress. The stress of unfolded protein accumulation has been implicated previously in low protein yields during heterologous protein expression. Here we describe the dynamics of the response to this stress, termed the unfolded protein response (UPR), during the expression of the single chain antibody 4-4-20 (scFv) in Saccharomyces cerevisiae. Expression of scFv decreased the growth rate of yeast cells whether the scFv was expressed from single-copy plasmids or integrated into the chromosome. However, the growth rates recovered at longer expression times, and surprisingly, the recovery occurred more quickly in the high-copy integration strains. The presence of a functional UPR pathway was necessary for a recovery of normal growth rates. During the growth inhibition, the UPR pathway appeared to be activated, resulting in decreased intracellular scFv levels and intermittent recovery of the chaperone BiP within the endoplasmic reticulum. Intracellular scFv was observed primarily in the endoplasmic reticulum, consistent with activation of the UPR pathway. Although the intracellular scFv levels dropped over the course of the expression, this was not a result of scFv secretion. A functional UPR pathway was necessary for the drop in intracellular scFv, suggesting that the decrease was a direct response of UPR activation. Taken together, these results suggest that control of heterologous gene expression to avoid UPR activation will result in higher production levels.