Effects Of Chaperones Research Articles

Quantification of differential efficacy of chemical chaperones in ameliorating solubilization and folding of zebrafish dihydrofolate reductase

Protein aggregation is a major hindrance in many in vivo and in vitro studies of proteins. It results in the formation of inclusion bodies and non-functional aggregates. Chemical chaperones also known as osmolytes which are accumulated during the stress conditions in the cells can influence the protein stability through various mechanisms. They act as osmoprotectants and contribute to the protein folding by enabling the protein to bury the backbone into the core of protein fold. In the current study, we observed the effect of chemical chaperones from four different classes on the stability and functionality of aggregation prone protein zebrafish dihydrofolate reductase (zDHFR). We also used UV–visible and circular dichroism (CD) spectroscopy to explore the protecting action of chemical chaperones on the structure and activity of zDHFR in vitro and in vivo conditions.

Effects of calcium-dependent molecular chaperones and endoplasmic reticulum in the amygdala in rats under single‑prolonged stress.

The purpose of the present study was to investigate the role of endoplasmic reticulum (ER)‑resident molecular chaperone proteins to identify whether these proteins were involved in post‑traumatic stress disorder (PTSD). The present study detected changes of calreticulin (CRT), calnexin (CNX) and ERp57 in the amygdala of rats, which may with aim of providing a novel insight into the modulation effect of amygdala in PTSD. Single‑prolonged stress (SPS) was applied to create the models of PTSD in rats. The expression levels of CRT, CNX and ERp57 were examined using immunohistochemistry or immunofluorescence, western blot analysis and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). The results showed that SPS induced significant changes in CRT, CNX and ERp57 expression levels. Furthermore, the expression levels of CRT, CNX and ERp57 were significantly upregulated when compared to that in the control group after SPS exposure by western blot analysis (P<0.05). RT‑qPCR analysis supported these results, indicating an upregulation of mRNA expression level. Taken together, the present findings suggest that SPS may induce changes to the expression of CRT, CNX and ERp57 in the amygdala of rats. The present study provides an insight into the effects of ER‑resident molecular chaperones in the amygdala participating in PTSD, and provides the experimental basis and a mechanism for the pathophysiology of PTSD.

NMR spectra of PB2 627, the RNA-binding domain in influenza A virus RNA polymerase that contains the pathogenicity factor lysine 627, and improvement of the spectra by small osmolytes

The influenza A virus, which has an RNA genome, requires RNA-dependent RNA polymerase for transcription and replication. The polymerase is comprised of the subunits PA, PB1, and PB2. The C-terminal RNA-binding domain in PB2 contains lysine 627 (PB2 627), which is associated with pathogenicity and host range. However, the structure and molecular mechanism of PB2 627 in solution remain obscure. Here, we investigated PB2 627 in solution by nuclear magnetic resonance (NMR) and detected inhomogeneity in the intensities of backbone amide proton signals due to local fluctuations in structure. To characterize the effects of chemical chaperones on spectral data and improve the data quality, we tested 20 different additives, including L-arginine L-glutamate salt, (L-arginine)2SO4, glycerol, β-octylglucoside, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, Na2SO4, 1,5-diaminopentane, 1,4-diaminobutane, trehalose, sucrose, glycine, trimethylamine N-oxide, β-alanine, L-α-alanine, hydroxyectoine, betaine, L-proline, and non-detergent sulfobetaine 195, 201, and 256. We evaluated the quality of the resulting spectra by calculating the standard deviation and average of the ratio of signal intensities to noise level of amide peaks, as well as the ratio of the standard deviation to the average. NMR-profile analysis revealed diverse effects of additives on the dynamic properties of PB2 627. Based on such criteria, we found that small osmolytes such as glycine and L-α-alanine reduced structural fluctuations and improved the quality of spectral data, which is likely to facilitate a detailed NMR-based structural analysis. The methodology developed here may also be more generally useful for evaluating the effects of chemical chaperones on the structural integrity of proteins.

Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real-time bioluminescence imaging.

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods.

Differential effects of chaperones on yeast prions: CURrent view

Endogenous yeast amyloids that control heritable traits and are frequently used as models for human amyloid diseases are termed yeast prions. Yeast prions, including the best studied ones ([PSI +] and [URE3]), propagate via intimate interactions with molecular chaperones. Different yeast prions exhibit differential responses to changes in levels, functionality or localization of the components of chaperone machinery. Here, we provide additional data confirming differential effects of chaperones (and specifically, Hsp40s) on yeast prions and summarize current knowledge of the mechanisms underlying chaperone specificities. Contrary to frequent statements in literature, overproduction of the Hsp104 chaperone antagonizes both [PSI +] and [URE3] prions, while overproduction of the Hsp70-Ssa1 chaperone antagonizes [URE3] prion only in some, but not in all strains. Recently, we demonstrated that the relocalization of a fraction of the Hsp40 chaperone Sis1 from the cytosol to the nucleus by the chaperone-sorting factor Cur1 exhibits opposite effects on [PSI +] and [URE3] prions. We suggest that the response of prions to changes in Sis1 localization represents a combination of the effects of Sis1 shortage on fragmentation of prion aggregates and on malpartition of prion aggregates during a cell division. Differences in sensitivity of prion fragmentation to Sis1 and in relative inputs of fragmentation and malpartition in prion propagation result in opposite effects of Sis1 relocalization on [PSI +] and [URE3].

Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding

The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the β-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA β-barrel domain, but is greater when full-length BamA is present, indicating that both the β-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro.

In silicoanalyses of essential interactions of iminosugars with the Hex A active site and evaluation of their pharmacological chaperone effects for Tay–Sachs disease

The affinity of a series of iminosugar-based inhibitors exhibiting various ring sizes toward Hex A and their essential interactions with the enzyme active site were investigated. All the Hex A-inhibiting iminosugars tested formed hydrogen bonds with Arg178, Asp322, Tyr421 and Glu462 and had the favorable cation-π interaction with Trp460. Among them, DMDP amide (6) proved to be the most potent competitive inhibitor with a Ki value of 0.041 μM. We analyzed the dynamic properties of both DMDP amide (6) and DNJNAc (1) in aqueous solution using molecular dynamics (MD) calculations; the distance of the interaction between Asp322 and 3-OH and Glu323 and 6-OH was important for stable interactions with Hex A, reducing fluctuations in the plasticity of the active site. DMDP amide (6) dose-dependently increased intracellular Hex A activity in the G269S mutant cells and restored Hex A activity up to approximately 43% of the wild type level; this effect clearly exceeded the border line treatment for Tay-Sachs disease, which is regarded as 10-15% of the wild type level. This is a significantly greater effect than that of pyrimethamine, which is currently in Phase 2 clinical trials. DMDP amide (6), therefore, represents a new promising pharmacological chaperone candidate for the treatment of Tay-Sachs disease.

Protein disulfide isomerases: Redox connections in and out of the endoplasmic reticulum

Protein disulfide isomerases are thiol oxidoreductase chaperones from thioredoxin superfamily. As redox folding catalysts from the endoplasmic reticulum (ER), their roles in ER-related redox homeostasis and signaling are well-studied. PDIA1 exerts thiol oxidation/reduction and isomerization, plus chaperone effects. Also, substantial evidence indicates that PDIs regulate thiol-disulfide switches in other cell locations such as cell surface and possibly cytosol. Subcellular PDI translocation routes remain unclear and seem Golgi-independent. The list of signaling and structural proteins reportedly regulated by PDIs keeps growing, via thiol switches involving oxidation, reduction and isomerization, S-(de)nytrosylation, (de)glutathyonylation and protein oligomerization. PDIA1 is required for agonist-triggered Nox NADPH oxidase activation and cell migration in vascular cells and macrophages, while PDIA1-dependent cytoskeletal regulation appears a converging pathway. Extracellularly, PDIs crucially regulate thiol redox signaling of thrombosis/platelet activation, e.g., integrins, and PDIA1 supports expansive caliber remodeling during injury repair via matrix/cytoskeletal organization. Some proteins display regulatory PDI-like motifs. PDI effects are orchestrated by expression levels or post-translational modifications. PDI is redox-sensitive, although probably not a mass-effect redox sensor due to kinetic constraints. Rather, the “all-in-one” organization of its peculiar redox/chaperone properties likely provide PDIs with precision and versatility in redox signaling, making them promising therapeutic targets.

Obesity-induced endoplasmic reticulum stress suppresses nuclear factor-Y expression.

Nuclear transcription factor Y (NF-Y) is an evolutionarily conserved transcription factor composed of three subunits, NF-YA, NF-YB, and NF-YC. NF-Y plays crucial roles in pre-adipocyte maintenance and/or commitment to adipogenesis. NF-YA dysfunction in adipocyte resulted in an age-dependent progressive loss of adipose tissue associated with metabolic complications. Endoplasmic reticulum (ER) stress has emerged as an important mediator in the pathogenesis of obesity. However, it is not known if NF-YA is involved in the ER stress-mediated pathogenesis of obesity. We first examined the effects of ER stress on the NF-YA expression in cultured 3T3-L1 adipocytes; then in ob/ob genetic obesity mice, we tested the effect of chemical chaperones alleviating ER stress on the expression levels of NF-YA. Subsequently, we inhibited the new mRNA synthesis using actinomycin D in 3T3-L1 cells to explore the mechanism modulating NF-YA expression. Finally, we evaluated the involvement of PPARg in the regulation of NF-YA expression by ER stress. We demonstrated that both obesity- and chemical chaperone -induced ER stress suppressed NF-YA expression and alleviation of ER stress by chemical chaperone could recover NF-YA expression in ob/ob mice. Moreover, we showed that ER stress suppressed NF-YA mRNA transcription through the involvement of peroxisome proliferator-activated receptor gamma (PPARg). Activation of PPARg ameliorates the ER stress-induced NF-YA suppression. Our findings may point to a possible role of NF-YA in stress conditions that occur in chronic obesity, ER stress might be involved in the pathogenesis of obesity through NF-YA depletion.

Effect of Chaperones on Prion Protein PrP23-98 Aggregation In Vitro.

Recent studies have indicated that PrP23-98, an N-terminal portion of PrP, polymerizes into amyloid-like and proteinase K (PK)-resistant aggregates in the presence of NADPH with copper ions [19], and then that CRT suppressed aggregation of PrP23-98 and also promoted solubilization of the aggregates [18]. As it is interesting to find out whether other chaperones can inhibit aggregation of PrP23-98 in vitro similar to CRT, this study was conducted to determine whether BiP, Grp94, PDI Grp58 and heat shock cognate protein70 (Hsc70) can inhibit aggregation of PrP23-98 in vitro. The present results indicated that Grp94 suppressed aggregation of PrP23-98, but that Grp94 could not mediate solubilization occurred in the aggregates in contrast to CRT. Other chaperons induced aggregation of PrP23-98 in the absence of NADPH.

Therapeutic Approaches Targeting Pathological Tau Aggregates

Neurodegenerative diseases characterized by the accumulation of tau aggregates are increasing in prevalence to epidemic-like levels and there is currently no effective treatment. For many years, the focus of tau-based research was on the fibrillar, neurofibrillary tangles. However, the compilation of evidence obtained from numerous laboratories in the past few years suggests that soluble intermediate aggregates-tau oligomers-are actually the most toxic protein species in disease. Thus, therapeutic agents that target oligomeric tau specifically may be the most effective routes for treatment. A great deal of progress has been made in the pre-clinical evaluation of a number of different anti-tau therapeutics. Upstream modulators of tau modifications have been evaluated and may provide some benefits, but likely will not be capable of eliminating toxic tau entirely. Protein chaperones capable of modulating the structure of tau and targeting it for degradation are another field of study, however, the broad effects of chaperones make side effects a concern. Thus, more specific agents capable of eliminating the most toxic species in disease are promising. Small molecules designed to inhibit aggregation, as well as immunotherapy with antibodies specific for toxic tau aggregates present the most advancement as potential treatments. The concerted effort across a number of groups to investigate potential mechanisms to inhibit tau toxicity represents great progress in the field and provides hope that effective treatments will be discovered.

DnaK-Dependent Accelerated Evolutionary Rate in Prokaryotes.

Many proteins depend on an interaction with molecular chaperones in order to fold into a functional tertiary structure. Previous studies showed that protein interaction with the GroEL/GroES chaperonine and Hsp90 chaperone can buffer the impact of slightly deleterious mutations in the protein sequence. This capacity of GroEL/GroES to prevent protein misfolding has been shown to accelerate the evolution of its client proteins. Whether other bacterial chaperones have a similar effect on their client proteins is currently unknown. Here, we study the impact of DnaK (Hsp70) chaperone on the evolution of its client proteins. Evolutionary parameters were derived from comparison of the Escherichia coli proteome to 1,808,565 orthologous proteins in 1,149 proteobacterial genomes. Our analysis reveals a significant positive correlation between protein binding frequency with DnaK and evolutionary rate. Proteins with high binding affinity to DnaK evolve on average 4.3-fold faster than proteins in the lowest binding affinity class at the genus resolution. Differences in evolutionary rates of DnaK interactor classes are still significant after adjusting for possible effects caused by protein expression level. Furthermore, we observe an additive effect of DnaK and GroEL chaperones on the evolutionary rates of their common interactors. Finally, we found pronounced similarities in the physicochemical profiles that characterize proteins belonging to DnaK and GroEL interactomes. Our results thus implicate DnaK-mediated folding as a major component in shaping protein evolutionary dynamics in bacteria and supply further evidence for the long-term manifestation of chaperone-mediated folding on genome evolution.

β-Casein will chaperone β-lactoglobulin during nanofibril assembly, but prefers familiar company at high concentrations

Bovine β-casein behaves as a chaperone at neutral pH, i.e., it inhibits aggregation of unfolded proteins, but chaperone effects have not been investigated at acidic pH. We examined chaperone effects during heating at pH 2 and 80 °C for mixtures of 1% (w/v) β-lactoglobulin (β-Lg) and β-Lg:β-casein molar ratios of 1:0.0625 to 1:1. These conditions hydrolyse β-Lg into peptides that self-assemble into amyloid-like nanofibrils. Hydrolysis rates in mixed solutions were unchanged from single-protein controls, according to sodium dodecyl sulphate polyacrylamide gel electrophoresis. At ratios ≥1:0.125, β-casein slowed fibril growth in a concentration-independent way, according to Thioflavin T fluorescence. We observed twisted irregular fibrils coexisting with long semiflexible fibrils when β-casein was heated with β-Lg, using transmission electron microscopy. We hypothesise that β-casein monomers and peptides can interact with growing β-Lg nanofibrils or assemble into micelles, with the latter pathway predominating at high β-casein concentrations.

Trigger Factor Reduces the Force Exerted on the Nascent Chain by a Cotranslationally Folding Protein

Cotranslational protein folding can generate pulling forces on the nascent chain that can affect the instantaneous translation rate and thereby possibly feed back on the folding process. Such feedback would represent a new way of coupling translation and folding, different from coupling based on, for example, codon usage. However, to date, we have carried out the experiments used to measure pulling forces generated by cotranslational protein folding either in reconstituted in vitro translation systems lacking chaperones, in ill-defined cell lysates, or in vivo; hence, the effects of chaperones on force generation by folding are unknown. Here, we have studied the cotranslational folding of dihydrofolate reductase (DHFR) in the absence and in the presence of the chaperones trigger factor (TF) and GroEL/ES. DHFR was tethered to the ribosome via a C-terminal linker of varying length, ending with the SecM translational arrest peptide that serves as an intrinsic force sensor reporting on the force generated on the nascent chain when DHFR folds. We find that DHFR folds into its native structure only when it has emerged fully outside the ribosome and that TF and GroEL alone substantially reduces the force generated on the nascent chain by the folding of DHFR, while GroEL/ES has no effect. TF therefore weakens the possible coupling between cotranslational folding and translation.

Chaperone-Mediated Mechanical Protein Folding at the Single Molecule Level

DnaK, DnaJ and GrpE heat shock proteins form the main conserved chaperone system in Escherichia coli. Such a complex chaperone system participates in the correct folding of emergent polypeptides in the ribosome to ensure that they reach their native structure, while preventing the aggregation of proteins under stress conditions in the cell. It is well known from bulk experiments that DnaK and DnaJ accomplish these functions by binding to both unfolded and partially unfolded protein substrates. However, the extended protein conformations that characterise the nascent proteins in the ribosome are rarely accessible by biochemistry denaturing experiments. Moreover, the detailed conformation of the substrates required for an optimal chaperone binding still remains controversial. Here we use single molecule force-clamp spectroscopy AFM to study the effect of molecular chaperones on the mechanical folding of individual proteins. This approach enables to monitor the individual folding trajectories from highly extended states, and to dissect the different conformational ensembles visited by the single protein during its folding pathway. Our experiments reveal that DnaJ binds to the unfolded state of Ubiquitin (which contains the consensus binding sequence GKQLEDG) with more affinity than to the molten globule, collapsed state. DnaJ binding blocks ubiquitin refolding by decreasing its folding efficiency by over 40%, thus highlighting its “holdase” functionality. By contrast, DnaK presents instead a higher affinity for the molten globule conformations. Crucially, both proteins, together with GrpE, readily increase the refolding of Ubiquitin by over 10%, within an ATP-mediated process. Altogether, our nanomechanical experiments demonstrate that the binding of different chaperones to a unique substrate is highly conformation-dependent, enabling us to postulate a kinetic scheme that underlies the synergetic functionality of a complex chaperone system, at the single molecule level.

Effect of molecular chaperones on aberrant protein oligomers in vitro: super-versus sub-stoichiometric chaperone concentrations.

Living systems protect themselves from aberrant proteins by a network of chaperones. We have tested in vitro the effects of different concentrations, ranging from 0 to 16 μm, of two molecular chaperones, namely αB-crystallin and clusterin, and an engineered monomeric variant of transthyretin (M-TTR), on the morphology and cytotoxicity of preformed toxic oligomers of HypF-N, which represent a useful model of misfolded protein aggregates. Using atomic force microscopy imaging and static light scattering analysis, all were found to bind HypF-N oligomers and increase the size of the aggregates, to an extent that correlates with chaperone concentration. SDS-PAGE profiles have shown that the large aggregates were predominantly composed of the HypF-N protein. ANS fluorescence measurements show that the chaperone-induced clustering of HypF-N oligomers does not change the overall solvent exposure of hydrophobic residues on the surface of the oligomers. αB-crystallin, clusterin and M-TTR can diminish the cytotoxic effects of the HypF-N oligomers at all chaperone concentration, as demonstrated by MTT reduction and Ca2+ influx measurements. The observation that the protective effect is primarily at all concentrations of chaperones, both when the increase in HypF-N aggregate size is minimal and large, emphasizes the efficiency and versatility of these protein molecules.

Chaperone-Assisted Soluble Expression of a Humanized Anti-EGFR ScFv Antibody in E. Coli.

Formation of inclusion bodies is a considerable obstacle threatening the advantages of E. coli expression system to serve as the most common and easiest system in recombinant protein production. To solve this problem, several strategies have been proposed among which application of molecular chaperones is of remarkable consideration. The aim of this study was to evaluate the effects of molecular chaperones on soluble expression of aggregation-prone humanized single chain antibody. To increase the solubility of a humanized single chain antibody (hscFv), different chaperone plasmids including PG-tf2 (GroES- GroEL- tig), ptf16 (tig) and pGro7 (GroES- GroEL) were co-expressed in BL21 cells containing pET-22b- hscFv construct. The solubility of recombinant hscFv was analyzed by SDS-PAGE. After purification of soluble hscFv by Ni-NTA column, the biological activity and cytotoxicity of the recombinant protein were tested by ELISA and MTT assay, respectively. SDS-PAGE analysis of the hscFv revealed that chaperone utility remarkably increased (up to 50%) the solubility of the protein. ELISA test and MTT assay analyses also confirmed the biological activity of the gained hscFv in reaction with A431 cells (OD value: 2.6) and inhibition of their proliferation, respectively. The results of this study revealed that co-expression of chaperones with hscFv leads to remarkable increase in the solubility of the recombinant hscFv, which could be of great consideration for large scale production of recombinant single chain antibodies.

Efficient Activation of Pathogenic ΔPhe501 Mutation in Monocarboxylate Transporter 8 by Chemical and Pharmacological Chaperones.

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transmembrane transporter expressed in many cell types, including neurons. Mutations that inactivate transport activity of MCT8 cause severe X-linked psychomotor retardation in male patients, a syndrome originally described as the Allan-Herndon-Dudley syndrome. Treatment options currently explored the focus on finding thyroid hormone-like compounds that bypass MCT8 and enter cells through different transporters. Because MCT8 is a multipass transmembrane protein, some pathogenic mutations affect membrane trafficking while potentially retaining some transporter activity. We explore here the effects of chemical and pharmacological chaperones on the expression and transport activity of the MCT8 mutant ΔPhe501. Dimethylsulfoxide, 4-phenylbutyric acid as well as its sodium salt, and the isoflavone genistein increase T3 uptake into MDCK1 cells stably transfected with mutant MCT8-ΔPhe501. We show that ΔPhe501 represents a temperature-sensitive mutant protein that is stabilized by the proteasome inhibitor MG132. 4-Phenylbutyrate has been used to stabilize ΔPhe508 mutant cystic fibrosis transmembrane conductance regulator protein and is in clinical use in patients with urea cycle defects. Genistein is enriched in soy and available as a nutritional supplement. It is effective in stabilizing MCT8-ΔPhe501 at 100 nM concentration. Expression of the L471P mutant is increased in response to phenylbutyrate, but T3 uptake activity is not induced, supporting the notion that the chaperone specifically increases membrane expression. Our findings suggest that certain pathogenic MCT8 mutants may be responsive to (co-)treatment with readily available compounds, which increase endogenous protein function.

Nucleotide Dependency of Endogenous Hsp90 Chaperone Protein Machinery and Its Association with Baculovirally Expressed Glucocorticoid Receptor

Hsp90 and hsp70 are essential eukaryotic chaperone proteins required for facilitating functional activity of mammalian transcription factors, including the glucocorticoid receptor (GR). Both chaperones exist in ATP‐ and ADP‐bound states, and understanding the relationship between nucleotide‐bound forms of hsp90 and hsp70, their affiliated cochaperones, and GR activation has significant pharmacological implications. The goal of this study was to investigate the extent to which hsp90 and hsp70 nucleotide interactions affect production of a GR‐hsp90 heterocomplex capable of steroid binding activity. Reticulocyte lysate (RL) containing high endogenous concentrations of hsp90 and hsp70 was incubated in the presence or absence of an ATP regenerating system and fractionated by size exclusion chromatography (SEC). ATP incubation did not appear to alter total global protein elution, as detected by A280 spectrophotometry. However, SDS‐PAGE and western blot analysis identified nucleotide‐associated alterations in hsp90 SEC elution profiles. In order to further compare nucleotide dependency and chaperone effects, full‐length GR, produced in baculovirally infected insect cells, was immunoadsorbed, stripped of endogenous chaperones, and reassembled into a heterocomplex with hsp90 ± ATP. SDS‐PAGE and western blotting showed hsp90 derived from RL−ATP samples were tightly bound to the GR, while hsp90 from RL+ATP failed to stay bound to the GR. The difference in SEC elution profiles and co‐immunoadsorption imply a notable role of nucleotide alterations in the hsp90 chaperone machinery and functional interactions with the GR.

High-level soluble expression of a bacterial N-acyl-d-glucosamine 2-epimerase in recombinant Escherichia coli

N-Acyl-d-glucosamine 2-epimerase (AGE) is an important enzyme for the biocatalytic synthesis of N-acetylneuraminic acid (Neu5Ac). Due to the wide range of biological applications of Neu5Ac and its derivatives, there has been great interest in its large-scale synthesis. Thus, suitable strategies for achieving high-level production of soluble AGE are needed. Several AGEs from various organisms have been recombinantly expressed in Escherichia coli. However, the soluble expression level was consistently low with an excessive formation of inclusion bodies. In this study, the effects of different solubility-enhancement tags, expression temperatures, chaperones and host strains on the soluble expression of the AGE from the freshwater cyanobacterium Anabaena variabilis ATCC 29413 (AvaAGE) were examined. The optimum combination of tag, expression temperature, co-expression of chaperones and host strain (His6-tag, 37°C, GroEL/GroES, E. coli BL21(DE3)) led to a 264-fold improvement of the volumetric epimerase activity, a measure of the soluble expression, compared to the starting conditions (His6-maltose-binding protein-tag, 20°C, without chaperones, E. coli BL21(DE3)). A maximum yield of 22.5mg isolated AvaAGE per liter shake flask culture was obtained.