General Molecular Chaperones Research Articles

Twin-arginine translocase component TatB performs folding quality control via a chaperone-like activity

The twin-arginine translocation (Tat) pathway involves an inbuilt quality control (QC) system that synchronizes the proofreading of substrate protein folding with lipid bilayer transport. However, the molecular details of this QC mechanism remain poorly understood. Here, we hypothesized that the conformational state of Tat substrates is directly sensed by the TatB component of the bacterial Tat translocase. In support of this hypothesis, several TatB variants were observed to form functional translocases in vivo that had compromised QC activity as evidenced by the uncharacteristic export of several misfolded protein substrates. These variants each possessed cytoplasmic membrane-extrinsic domains that were either truncated or mutated in the vicinity of a conserved, highly flexible α-helical domain. In vitro folding experiments revealed that the TatB membrane-extrinsic domain behaved like a general molecular chaperone, transiently binding to highly structured, partially unfolded intermediates of a model protein, citrate synthase, in a manner that prevented its irreversible aggregation and stabilized the active species. Collectively, these results suggest that the Tat translocase may use chaperone-like client recognition to monitor the conformational status of its substrates.

Molecular chaperones and G protein-coupled receptor maturation and pharmacology

G protein-coupled receptors (GPCRs) are highly conserved versatile signaling molecules located at the plasma membrane that respond to diverse extracellular signals. They regulate almost all physiological processes in the vertebrates. About 35% of current drugs target these receptors. Mutations in these genes have been identified as causes of numerous diseases. The seven transmembrane domain structure of GPCRs implies that the folding of these transmembrane proteins is extremely complicated and difficult. Indeed, many wild type GPCRs are not folded optimally. The most common defect in genetic diseases caused by GPCR mutations is misfolding and failure to reach the plasma membrane where it functions. General molecular chaperones aid the folding of all proteins, including GPCRs, by preventing aggregation, promoting folding and disaggregating small aggregates. Some GPCRs need additional receptor-specific chaperones to assist their folding. Many of these receptor-specific chaperones interact with additional receptors and alter receptor pharmacology, expanding the understanding of these chaperone proteins.

Unusual pairing between assistants: Interaction of the twin-arginine system-specific chaperone DmsD with the chaperonin GroEL

DmsD is a system-specific chaperone that mediates the biogenesis and maturation of DMSO reductase in Escherichia coli. It is required for DmsAB holoenzyme formation and its targeting to the cytoplasmic membrane for translocation by the twin-arginine translocase. Previous studies suggested that DmsD also interacts with general molecular chaperones to assist in folding of the reductase subunits. Here, the interaction between DmsD and GroEL was further characterized to understand the role of GroEL in DMSO reductase maturation. The inherently weak interaction between the two was strengthened in vivo under growth conditions that induce DMSO reductase expression, and the DmsD–GroEL complex showed negligible change in hydrodynamic diameter by dynamic light scattering when cross-linked. Mapping the cross-linked sites on DmsD shows that the GroEL binding site is in close proximity to the previously characterized DmsA leader binding site. These findings support a role of GroEL in DMSO reductase maturation that likely involves its chaperonin function for assisting in folding of the DmsA preprotein.

Molecular Chaperone Function for Myocilin

Myocilin is thought to be a stress response protein, but its exact molecular functions have not been established. Studies were conducted to see whether myocilin can act as a general molecular chaperone. Myocilin was isolated and purified from porcine trabecular meshwork (TM) cell culture media. Its ability to protect citrate synthase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the restriction endonuclease DrdI from thermal inactivation was evaluated. Light scattering was used to evaluate thermally induced aggregation of citrate synthase. Myocilin induction was assessed after exposure of TM cells to several types of stress treatments. Levels of extracellular myocilin expressed by TM cells were increased in response to mechanical stretch, heat shock, TNFα, or IL-1α. Myocilin protected citrate synthase activity against thermal inactivation for 5 minutes at 55°C in a concentration-dependent manner, with nearly full protection of 1.5 μM citrate synthase in the presence of 650 nM myocilin. Myocilin significantly reduced thermal aggregation of citrate synthase to levels 36% to 44% of control levels. Myocilin also protected GAPDH from thermal inactivation for 10 minutes at 45°C. Myocilin at 18 nM was more effective than 1 μM bovine serum albumin at protecting DrdI from thermal inactivation. Myocilin is induced in response to several cellular stresses and displays general molecular chaperone activity by protecting DrdI, citrate synthase, and GAPDH from thermal inactivation. Myocilin also suppresses the thermal aggregation of citrate synthase. One function of myocilin may be to serve as a molecular chaperone.

The precursor to the germ cell-specific PCSK4 proteinase is inefficiently activated in transfected somatic cells: evidence of interaction with the BiP chaperone

Proprotein convertase subtilisin/kexin type 4 (PCSK4), also known as proprotein convertase 4 (PC4), is a serine endoproteinase primarily expressed in testicular germ cells and in sperm. Inactivation of its gene in mouse causes male infertility. From studies of the biosynthesis of PCSK3/furin, its closest relative, it has been inferred that PCSK4 is synthesised in the endoplasmic reticulum as a zymogen; that it is rapidly matured by autocatalytic cleavage between the prodomain and the catalytic domain; that the cleaved prodomain remains attached to the mature enzyme; and that the enzyme is finally activated by the removal of the prodomain peptides following a secondary cleavage within the prodomain. In this study, we used human embryonic kidney 293 (HEK293) cells to study the biosynthesis of rat or human PCSK4. Our results show that the bulk of PCSK4 remains as an intracellular zymogen, presumably trapped in the endoplasmic reticulum, where it interacts with the general molecular chaperone glucose-regulated protein 78/Immunoglobulin heavy-chain binding protein (GRP78/BiP). These data suggest that, unlike other members of the convertase family, proPCSK4 cannot efficiently self-activate in somatic cells. These cells may lack the intracellular environment and the interacting molecules specific to testicular germ cells where this enzyme is normally expressed.

DmsD, a Tat system specific chaperone, interacts with other general chaperones and proteins involved in the molybdenum cofactor biosynthesis

Many bacterial oxidoreductases depend on the Tat translocase for correct cell localization. Substrates for the Tat translocase possess twin-arginine leaders. System specific chaperones or redox enzyme maturation proteins (REMPs) are a group of proteins implicated in oxidoreductase maturation. DmsD is a REMP discovered in Escherichia coli, which interacts with the twin-arginine leader sequence of DmsA, the catalytic subunit of DMSO reductase. In this study, we identified several potential interacting partners of DmsD by using several in vitro protein–protein interaction screening approaches, including affinity chromatography, co-precipitation, and cross-linking. Candidate hits from these in vitro findings were analyzed by in vivo methods of bacterial two-hybrid (BACTH) and bimolecular fluorescence complementation (BiFC). From these data, DmsD was confirmed to interact with the general molecular chaperones DnaK, DnaJ, GrpE, GroEL, Tig and Ef-Tu. In addition, DmsD was also found to interact with proteins involved in the molybdenum cofactor biosynthesis pathway. Our data suggests that DmsD may play a role as a “node” in escorting its substrate through a cascade of chaperone assisted protein-folding maturation events.

An Essential Role for the DnaK Molecular Chaperone in Stabilizing Over-expressed Substrate Proteins of the Bacterial Twin-arginine Translocation Pathway

All secreted proteins in Escherichia coli must be maintained in an export-competent state before translocation across the inner membrane. In the case of the Sec pathway, this function is carried out by the dedicated SecB chaperone and the general chaperones DnaK-DnaJ-GrpE and GroEL-GroES, whose job collectively is to render substrate proteins partially or entirely unfolded before engagement of the translocon. To determine whether these or other general molecular chaperones are similarly involved in the translocation of folded proteins through the twin-arginine translocation (Tat) system, we screened a collection of E. coli mutant strains for their ability to transport a green fluorescent protein (GFP) reporter through the Tat pathway. We found that the molecular chaperone DnaK was essential for cytoplasmic stability of GFP bearing an N-terminal Tat signal peptide, as well as for numerous other recombinantly expressed endogenous and heterologous Tat substrates. Interestingly, the stability conferred by DnaK did not require a fully functional Tat signal as substrates bearing translocation defective twin lysine substitutions in the consensus Tat motif were equally unstable in the absence of DnaK. These findings were corroborated by crosslinking experiments that revealed an in vivo association between DnaK and a truncated version of the Tat substrate trimethylamine N-oxide reductase (TorA502) bearing an RR or a KK signal peptide. Since TorA502 lacks nine molybdo-cofactor ligands essential for cofactor attachment, the involvement of DnaK is apparently independent of cofactor acquisition. Finally, we show that the stabilizing effects of DnaK can be exploited to increase the expression and translocation of Tat substrates under conditions where the substrate production level exceeds the capacity of the Tat translocase. This latter observation is expected to have important consequences for the use of the Tat system in biotechnology applications where high levels of periplasmic expression are desirable.

Structural and Functional Conversion of Molecular Chaperone ClpB from the Gram-Positive Halophilic Lactic Acid Bacterium Tetragenococcus halophilus Mediated by ATP and Stress

In this study, we report the purification, initial structural characterization, and functional analysis of the molecular chaperone ClpB from the gram-positive, halophilic lactic acid bacterium Tetragenococcus halophilus. A recombinant T. halophilus ClpB (ClpB(Tha)) was overexpressed in Escherichia coli and purified by affinity chromatography, hydroxyapatite chromatography, and gel filtration chromatography. As demonstrated by gel filtration chromatography, chemical cross-linking with glutaraldehyde, and electron microscopy, ClpB(Tha) forms a homohexameric single-ring structure in the presence of ATP under nonstress conditions. However, under stress conditions, such as high-temperature (>45 degrees C) and high-salt concentrations (>1 M KCl), it dissociated into dimers and monomers, regardless of the presence of ATP. The hexameric ClpB(Tha) reactivated heat-aggregated proteins dependent upon the DnaK system from T. halophilus (KJE(Tha)) and ATP. Interestingly, the mixture of dimer and monomer ClpB(Tha), which was formed under stress conditions, protected substrate proteins from thermal inactivation and aggregation in a manner similar to those of general molecular chaperones. From these results, we hypothesize that ClpB(Tha) forms dimers and monomers to function as a holding chaperone under stress conditions, whereas it forms a hexamer ring to function as a disaggregating chaperone in cooperation with KJE(Tha) and ATP under poststress conditions.

Inhibition of GR-mediated transcription by p23 requires interaction with Hsp90

p23 is a regulatory co-chaperone of heat shock protein (Hsp) 90, but can also act as a general molecular chaperone by itself. Using novel point mutations of p23 that disrupt its interaction with Hsp90 we found its co-chaperone function to be required for its inhibitory effect on glucocorticoid receptor (GR). The C-terminal region of p23, which is required for its chaperone activity, is dispensable for inhibition of GR. Importantly, similar results were obtained with a constitutively active GR. Thus, the action of p23 on the nuclear stage of GR regulation requires its Hsp90 co-chaperone function, but not its chaperone activity.

Maltose-binding protein as a solubility enhancer.

A major impediment to the production of recombinant proteins in Escherichia coli is their tendency to accumulate in the form of insoluble and biologically inactive aggregates known as inclusion bodies. Although it is sometimes possible to convert aggregated material into native, biologically-active protein, this is a time consuming, labor-intensive, costly, and uncertain undertaking (1). Consequently, many tricks have been employed in an effort to circumvent the formation of inclusion bodies (2). One approach that shows considerable promise is to exploit the innate ability of certain proteins to enhance the solubility of their fusion partners. Although it was originally thought that virtually any highly soluble protein could function as a general solubilizing agent, this has not turned out to be the case. In a direct comparison with glutathione S-transferase (GST) and thioredoxin, maltose-binding protein (MBP) was decidedly superior at solubilizing a diverse collection of aggregation-prone passenger proteins (3). Moreover, some of these proteins were able to fold into their biologically active conformations when fused to MBP. It is not entirely clear why MBP is such a spectacular solubilizing agent, but there is some evidence to suggest that it may be able to function as a general molecular chaperone in the context of a fusion protein by temporarily sequestering aggregation-prone folding intermediates of its fusion partners and preventing their self association (3–6).

Overexpression of DsbC and DsbG markedly improves soluble and functional expression of single-chain Fv antibodies in Escherichia coli

Single-chain Fv antibodies (scFv), a group of reconstructed molecules with several disulfide bonds, are prone to aggregate as inclusion bodies, the insoluble species of natural proteins, when expressed in Escherichia coli, especially at high level. Recovery of functionally active products from inclusion bodies is onerous and ineffective. We have increased the soluble and functional scFv yields by fusing either DsbC or DsbG, two E. coli disulfide isomerases with general chaperone function, to scFvs. Compared to the totally insoluble inclusion bodies of scFvs expressed separately, more than half of each fusion protein DsbC-scFv or DsbG-scFv was soluble, according to SDS–PAGE analysis. The more effective solubility was obtained when the fused protein DsbG-scFv was co-expressed simultaneously with DsbC under the same promoter. Under this condition, the soluble portion of DsbG-scFv increased from about 50% to 90% measured by scanning SDS–PAGE gel. Co-expression of DsbC can change fusion protein CBD-scFv from totally insoluble when expressed in E. coli separately to a considerable portion of soluble CBD-scFv. Antigen-binding activity assay showed that scFvs retained full affinity to specific antigens. We also determined that general molecular chaperones GroEL and GroES had no effects on the solubility of scFvs when co-expressed with scFv in E. coli. We propose that the correct formation of disulfide bonds in scFvs is the crucial factor responsible for solubility of scFvs.

Single amino acid substitutions on the surface of Escherichia coli maltose-binding protein can have a profound impact on the solubility of fusion proteins.

Proteins are commonly fused to Escherichia coli maltose-binding protein (MBP) to enhance their yield and facilitate their purification. In addition, the stability and solubility of a passenger protein can often be improved by fusing it to MBP. In a previous comparison with two other highly soluble fusion partners, MBP was decidedly superior at promoting the solubility of a range of aggregation-prone proteins. To explain this observation, we proposed that MBP could function as a general molecular chaperone in the context of a fusion protein by binding to aggregation-prone folding intermediates of passenger proteins and preventing their self-association. The ligand-binding cleft in MBP was considered a likely site for peptide binding because of its hydrophobic nature. We tested this hypothesis by systematically replacing hydrophobic amino acid side chains in and around the cleft with glutamic acid. None of these mutations affected the yield or solubility of MBP in its unfused state. Each MBP was then tested for its ability to promote solubility when fused to three passenger proteins: green fluorescent protein, p16, and E6. Mutations within the maltose-binding cleft (W62E, A63E, Y155E, W230E, and W340E) had little or no effect on the solubility of the fusion proteins. In contrast, three mutations near one end of the cleft (W232E, Y242E, and I317E) dramatically reduced the solubility of the same fusion proteins. The mutations with the most profound effect on solubility were shown to reduce the global stability of MBP.

Functional genomic approaches to understanding molecular chaperones and stress responses

This chapter provides a few key examples that underscore how characterization of proteins identified solely on the basis of their status as conserved targets of the heat shock response has led to insights into the cell biology of protein synthesis, folding, and degradation. In particular, this strategy has allowed the discovery of new general molecular chaperones, a connection between the cellular chaperone and degradation machinery, and, more recently, an unexpected relationship between ribosome metabolism and the heat shock response. The chapter concentrates on lessons learned from the application of genomic array technology to a specific folding stress response, the unfolded protein response (UPR), which is critical for maintaining the integrity of protein folding in the endoplasmic reticulum (ER). In addition, the chapter also focuses on the characterization of the different cellular processes induced by the UPR and speculates on how these various processes might act in a coherent way to aid ER folding.

A Temperature-Dependent Switch from Chaperone to Protease in a Widely Conserved Heat Shock Protein

Misfolding or unfolding of polypeptides can occur as a consequence of environmental stress and spontaneous mutation. The abundance of general chaperones and proteases suggests that cells distinguish between proteins that can be refolded and “hopeless” cases fated to enter the proteolytic pathway. The mechanisms controlling this key metabolic decision are not well understood. We show here that the widely conserved heat shock protein DegP (HtrA) has both general molecular chaperone and proteolytic activities. The chaperone function dominates at low temperatures, while the proteolytic activity is present at elevated temperatures. These results show that a single cellular factor can switch between two key pathways, controlling protein stability and turnover. Implications of this finding for intracellular protein metabolism are discussed.

Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.

Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation-prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners--maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX)--to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.

The 90 kDa heat-shock protein (hsp90) modulates the binding of the oestrogen receptor to its cognate DNA.

The role of heat-shock protein 90 (hsp90) in the regulation of the oestrogen receptor (ER) function is less well understood than for other steroid-hormone receptors because hsp90 is not involved in the stabilization or induction of a high-affinity ligand-binding state of ER nor in the inhibition of receptor dimerization. Electrophoretic mobility-shift assays, using purified ER and hsp90, were employed to investigate directly the effect of hsp90 on the ability of ER to bind to the oestrogen-response element (ERE) from the vitellogenin A2 gene. Contrary to models in which hsp90 binds to and passively inactivates steroid-hormone receptors, our studies show that the binding of ER to ERE is inversely dependent on the relative concentration of hsp90. Exposure of purified ER-hsp90 complexes to ERE led to the dissociation of hsp90 and concomitant specific binding of ER to ERE. We demonstrate that the amount of ER-ERE complex decreased with increasing concentrations of hsp90. Furthermore hsp90 dissociated preformed high-affinity ER-ERE complexes. Kinetic dissociation experiments indicate the hsp90 acts in a dynamic and specific process rather than by simple trapping of ER owing to its inherent off-rate. The receptor released from the ERE-bound state by hsp90 was recovered associated with hsp90 and was able to rebind to ERE. These results indicate that hsp90 does not suppress ER function merely by steric hindrance. On the basis of these results and others, we propose that, in vivo, hsp90 may play a dual role in ER function: (i) at a physiological temperature, hsp90 stabilizes an active form of the receptor in accordance with its general molecular chaperone role; (ii) at elevated temperatures or under other environmental stress, the increased cellular concentration of hsp90 negatively interferes with ER-dependent transcription, in accordance with the inhibition of gene transcription attributed to hsp90 after heat shock.

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross-linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross-linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion-specific and general molecular chaperone respectively, early in protein synthesis.