The Escherichia coli DjlA and CbpA proteins can substitute for DnaJ in DnaK-mediated protein disaggregation

The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37 degrees C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42 degrees C.     

Effect of molecular chaperones on the soluble expression of alginate lyase inE. coli

When the alginate lyase gene (aly) fromPseudoalteromonas elyakovii was expressed inE. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase inE. coli, we constructed plasmids designed to permit the coexpression ofaly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression ofaly with the Dnak/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration ofl-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.     

Effect of null mutations in dnaK and dnaJ genes on conjugational DNA transfer,proteolysis and novobiocin susceptibility of Escherichia coli

Escherichia coli null dnaJ and dnaKdnaJ mutants, when introduced to Hfr donor, impair its ability to DNA transfer during conjugation. The additive effect of both mutations was shown. Lack of DnaK and DnaJ chaperones also decrease the extent of proteolysis in mutant strains. This effect is seen only at 42 degrees C. The influence of double dnaKdnaJ deletion but not single dnaJ deletion on novobiocin susceptibility was also demonstrated.     

Cytosolic protein quality control system--the role of molecular chaperones in the biology of neurodegenerative diseases

In this article we describe the role of molecular chaperones and cellular proteases in the cytosolic protein quality control system that controls and regulates in all living organisms folding status of proteins and their proper function. Thanks to cooperative action of molecular chaperones and proteases the acumulation of misfolded proteins in the cytosol is limited. In particular, the links between chaperones to protein degradation and the role of molecular chaperones in the biology of neurodegnerative diseases are discussed.     

Monitoring protein conformation along the pathway of chaperonin-assisted folding

The GroEL/GroES chaperonin system mediates protein folding in the bacterial cytosol. Newly synthesized proteins reach GroEL via transfer from upstream chaperones such as DnaK/DnaJ (Hsp70). Here we employed single molecule and ensemble FRET to monitor the conformational transitions of a model substrate as it proceeds along this chaperone pathway. We find that DnaK/DnaJ stabilizes the protein in collapsed states that fold exceedingly slowly. Transfer to GroEL results in unfolding, with a fraction of molecules reaching locally highly expanded conformations. ATP-induced domain movements in GroEL cause transient further unfolding and rapid mobilization of protein segments with moderate hydrophobicity, allowing partial compaction on the GroEL surface. The more hydrophobic regions are released upon subsequent protein encapsulation in the central GroEL cavity by GroES, completing compaction and allowing rapid folding. Segmental chain release and compaction may be important in avoiding misfolding by proteins that fail to fold efficiently through spontaneous hydrophobic collapse.     

Lon Protease Quality Control of Presecretory Proteins in Escherichia coli and Its Dependence on the SecB and DnaJ (Hsp40) Chaperones

Various environmental insults result in irreversible damage to proteins and protein complexes. To cope, cells have evolved dedicated protein quality control mechanisms involving molecular chaperones and proteases. Here, we provide both genetic and biochemical evidence that the Lon protease and the SecB and DnaJ/Hsp40 chaperones are involved in the quality control of presecretory proteins in Escherichia coli. We showed that mutations in the lon gene alleviate the cold-sensitive phenotype of a secB mutant. Such suppression was not observed with either clpP or clpQ protease mutants. In comparison to the respective single mutants, the double secB lon mutant strongly accumulates aggregates of SecB substrates at physiological temperatures, suggesting that the chaperone and the protease share substrates. These observations were extended in vitro by showing that the main substrates identified in secB lon aggregates, namely proOmpF and proOmpC, are highly sensitive to specific degradation by Lon. In contrast, both substrates are significantly protected from Lon degradation by SecB. Interestingly, the chaperone DnaJ by itself protects substrates better from Lon degradation than SecB or the complete DnaK/DnaJ/GrpE chaperone machinery. In agreement with this finding, a DnaJ mutant protein that does not functionally interact in vivo with DnaK efficiently suppresses the SecB cold-sensitive phenotype, highlighting the role of DnaJ in assisting presecretory proteins. Taken together, our data suggest that when the Sec secretion pathway is compromised, a pool of presecretory proteins is transiently maintained in a translocation-competent state and, thus, protected from Lon degradation by either the SecB or DnaJ chaperones.     

From the cradle to the grave: molecular chaperones that may choose between folding and degradation

Molecular chaperones are known to facilitate cellular protein folding. They bind non-native proteins and orchestrate the folding process in conjunction with regulatory cofactors that modulate the affinity of the chaperone for its substrate. However, not every attempt to fold a protein is successful and chaperones can direct misfolded proteins to the cellular degradation machinery for destruction. Protein quality control thus appears to involve close cooperation between molecular chaperones and energy-dependent proteases. Molecular mechanisms underlying this interplay have been largely enigmatic so far. Here we present a novel concept for the regulation of the eukaryotic Hsp70 and Hsp90 chaperone systems during protein folding and protein degradation.     

Roles of molecular chaperones in endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD)

Secreted proteins are synthesized at the endoplasmic reticulum (ER), and a quality control mechanism in the ER is essential to maintain secretory pathway homeostasis. Newly synthesized soluble and integral membrane secreted proteins fold into their native conformations with the aid of ER molecular chaperones before they are transported to post-ER compartments. However, terminally mis-folded proteins may be retained in the ER and degraded by a process called ER-associated degradation (ERAD). Recent studies using yeast have shown that molecular chaperones both in the ER and in the cytosol play key roles during the ERAD of mis-folded proteins. One important role for chaperones during ERAD is to prevent substrate protein aggregation. Substrate selection is another important role for molecular chaperones during ERAD.     

Secreted phosphatase activity induced by dimethyl sulfoxide in Herpetomonas samuelpessoai

The secreted form of mouse meprin A is a homooligomer of meprin alpha subunits that contain a prosequence, a catalytic domain, and three domains designated as MAM (meprin, A5 protein, receptor protein-tyrosine phosphatase mu), MATH (meprin and TRAF homology), and AM (AfterMath). Previous studies indicated that wild-type mouse meprin alpha is predominantly a secreted protein, while the MAM deletion mutant (DeltaMAM) is degraded intracellularly. The work herein indicates that the DeltaMAM mutant is ubiquitinated and degraded via the proteasomal pathway. Both wild-type meprin alpha and the DeltaMAM mutant interact with the molecular chaperones calnexin and calreticulin in the endoplasmic reticulum. The interactions of the chaperones with the DeltaMAM mutant were significantly prolonged in the presence of lactacystin, a specific inhibitor of the proteasome, whereas those with the wild type were not affected by this inhibitor. Trimming of the Asn-linked core oligosaccharides of meprin subunits was required for interactions with the chaperones. The data indicated that folding of the wild-type protein was accelerated by chaperones, whereas the rate of dimerization was unaffected. Thus, calnexin and calreticulin are intimately involved in the correct folding and transport of meprin to the plasma membrane, as well as in retrograde transport of the DeltaMAM mutant to the ubiquitin-dependent proteasomal degradative pathway in the cytosol.     

The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species.

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.     

The C terminus of sigma(32) is not essential for degradation by FtsH

A key step in the regulation of heat shock genes in Escherichia coli is the stress-dependent degradation of the heat shock promoter-specific sigma(32) subunit of RNA polymerase by the AAA protease, FtsH. Previous studies implicated the C termini of protein substrates, including sigma(32), as degradation signals for AAA proteases. We investigated the role of the C terminus of sigma(32) in FtsH-dependent degradation by analysis of C-terminally truncated sigma(32) mutant proteins. Deletion of the 5, 11, 15, and 21 C-terminal residues of sigma(32) did not affect degradation in vivo or in vitro. Furthermore, a peptide comprising the C-terminal 21 residues of sigma(32) was not degraded by FtsH in vitro and thus did not serve as a recognition sequence for the protease, while an unrelated peptide of similar length was efficiently degraded. The truncated sigma(32) mutant proteins remained capable of associating with DnaK and DnaJ in vitro but showed intermediate (5-amino-acid deletion) and strong (11-, 15-, and 21-amino-acid deletions) defects in association with RNA polymerase in vitro and biological activity in vivo. These results indicate an important role for the C terminus of sigma(32) in RNA polymerase binding but no essential role for FtsH-dependent degradation and association of chaperones.     

Degradation of mutant proteins, underlying "loss of function" phenotypes, plays a major role in genetic disease

Many Mendelian monogenic disorders are caused by loss of the function of a single protein. This can result from rapid degradation of the mutant protein by cellular proteases, which reduces the steady-state concentration of the protein within the cell. The susceptibility of a protein to such proteolytic breakdown depends upon its kinetics of monomer folding and oligomer assembly and upon the intrinsic (thermodynamic) stability of its functional native-state conformation. Other cellular proteins, notably molecular chaperones, promote correct protein folding and assembly and thus provide some protection against degradation. An accumulation of recent evidence indicates that premature or accelerated degradation of mutant proteins, provoked by aberrations in their conformation, occurs in various subcellular compartments and represents a significant and prevalent pathogenic mechanism underlying genetic diseases. Inter-individual variability in proteolytic and folding systems can in part explain why "simple monogenic diseases" often display inconsistent genotype-phenotype correlations which show these disorders to be in reality quite complex. Protein folding and degradation may also be modulated artificially using exogenous small molecules. The identification or design of compounds which can interact specifically with particular target proteins, and which in so doing can exert beneficial effects on protein folding, assembly and/or stability, is beginning to open up a new and remarkably promising avenue for the treatment of diverse genetic disorders.     

Chaperone-mediated reduction of RepA dimerization is associated with RepA conformational change

RepA, the initiator protein of plasmid P1, binds to multiple sites (iterons) in the origin. The binding normally requires participation of chaperones, DnaJ, DnaK and GrpE. When purified, RepA appears dimeric and is inactive in iteron binding. On reaction with chaperones, a species active in iteron binding is formed and found to be monomeric. To test whether the chaperones can reduce dimerization, RepA was used to replace the dimerization domain of the λ repressor. The hybrid protein repressed the λ operator efficiently, indicating that RepA can dimerize in vivo . A further increase in repressor activity was seen in dnaJ mutant cells. These results are consistent with a chaperone-mediated reduction of RepA dimerization. We also found that RepA mutants defective in dimerization still depend on DnaJ for iteron binding. Conversely, RepA mutants that no longer require chaperones for iteron binding remain dimerization proficient. These results indicate that the chaperone dependence of RepA activity is not solely owing to RepA dimerization. Our results are most simply explained by a chaperone-mediated conformational change in RepA protomer that activates iteron binding. This conformational change also results in reduced RepA dimerization.     

Bacterial chaperones increase the production of soluble human TNF-alpha in Escherichia coli

Bacterial chaperones were coexpressed to enhance the production of soluble human TNF-alpha and its low-toxicity mutant in Escherichia coli. In the absence of chaperones, about 65% of wild-type TNF and 35% of mutant TNF having a deletion of N-terminal 7 amino acids and substitutions of Leu29'Ser, Ser52;Ile and Tyr56'Phe, were produced as a soluble form. In the presence of overproduced chaperones, most of wild-type and mutant TNF (>95%) were produced as a soluble form, indicating that GroEL and GroES chaperones promote folding and assembly of trimeric TNF-alpha. Bacterial chaperones could be useful in the production of TNF-alpha and its variants as well as other proteins with biological importance.     

Involvement of the molecular chaperone Ydj1 in the ubiquitin-dependent degradation of short-lived and abnormal proteins in Saccharomyces cerevisiae.

In Escherichia coli and mitochondria, the molecular chaperone DnaJ is required not only for protein folding but also for selective degradation of certain abnormal polypeptides. Here we demonstrate that in the yeast cytosol, the homologous chaperone Ydj1 is also required for ubiquitin-dependent degradation of certain abnormal proteins. The temperature-sensitive ydj1-151 mutant showed a large defect in the overall breakdown of short-lived cell proteins and abnormal polypeptides containing amino acid analogs, especially at 38 degrees C. By contrast, the degradation of long-lived cell proteins, which is independent of ubiquitin, was not altered nor was cell growth affected. The inactivation of Ydj1 markedly reduced the rapid, ubiquitin-dependent breakdown of certain beta-galactosidase (beta-gal) fusion polypeptides. Although degradation of N-end rule substrates (arginine-beta-gal and leucine-beta-gal) and the B-type cyclin Clb5-beta-gal occurred normally, degradation of the abnormal polypeptide ubiquitin-proline-beta-gal (Ub-P-beta-gal) and that of the short-lived normal protein Gcn4 were inhibited. As a consequence of reduced degradation of Ub-P-beta-gal, the beta-gal activity was four to five times higher in temperature-sensitive ydj1-151 mutant cells than in wild-type cells; thus, the folding and assembly of this enzyme do not require Ydj1 function. In wild-type cells, but not in ydj1-151 mutant cells, this chaperone is associated with the short-lived substrate Ub-P-beta-gal and not with stable beta-gal constructs. Furthermore, in the ydj1-151 mutant, the ubiquitination of Ub-P-beta-gal was blocked and the total level of ubiquitinated protein in the cell was reduced. Thus, Ydj1 is essential for the ubiquitin-dependent degradation of certain proteins. This chaperone may facilitate the recognition of unfolded proteins or serve as a cofactor for certain ubiquitin-ligating enzymes.     

Efficient production of native actin upon translation in a bacterial lysate supplemented with the eukaryotic chaperonin TRiC

Recombinant expression of actin in bacteria results in non-native species that aggregate into inclusion bodies. Actin is a folding substrate of TRiC, the chaperonin of the eukaryotic cytosol. By employing bacterial in vitro translation lysates supplemented with purified chaperones, we have found that TRiC is the only eukaryotic chaperone necessary for correct folding of newly translated actin. The actin thus produced binds deoxyribonuclease I and polymerizes into filaments, hallmarks of its native state. In contrast to its rapid folding in the eukaryotic cytosol, actin translated in TRiC-supplemented bacterial lysate folds with slower kinetics, resembling the kinetics upon refolding from denaturant. Lysate supplementation with the bacterial chaperonin GroEL/ES or the DnaK/DnaJ/GrpE chaperones leads to prevention of actin aggregation, yet fails to support its correct folding. This combination of in vitro bacterial translation and TRiC-assisted folding allows a detailed analysis of the mechanisms necessary for efficient actin folding in vivo. In addition, it provides a robust alternative for the production of substantial amounts of eukaryotic proteins that otherwise misfold or lead to cellular toxicity upon expression in heterologous hosts.     

The role of DnaK/DnaJ and GroEL/GroES systems in the removal of endogenous proteins aggregated by heat-shock from Escherichia coli cells

The submission of Escherichia coli cells to heat-shock (45 degrees C, 15 min) caused the intracellular aggregation of endogenous proteins. In the wt cells the aggregates (the S fraction) disappeared 10 min after transfer to 37 degrees C. In contrast, the S fraction in the dnaK and dnaJ mutant strains was stable during approximately one generation time (45 min). This demonstrated that neither the renaturation nor the degradation of the denatured proteins was possible in the absence of DnaK and DnaJ. The groEL44 and groES619 mutations stabilised the aggregates to a lesser extent. It was shown by the use of cloned genes, dnaK/dnaJ or groEL/groES, producing the corresponding proteins in about 4-fold excess, that the appearance of the S fraction in the wt strain resulted from a transiently insufficient supply of the heat-shock proteins. Overproduction of the GroEL/GroES proteins in dnaK756 or dnaJ259 background prevented the aggregation, however, overproduction of the DnaK/DnaJ proteins did not prevent the aggregation in the groEL44 or groES619 mutant cells although it accelerated the disappearance of the aggregates. The properties of the aggregated proteins are discussed from the point of view of their competence to renaturation/degradation by the heat-shock system.     

Differential degradation of variant medium-chain acyl-CoA dehydrogenase by the protein quality control proteases Lon and ClpXP

The coordinated activities of chaperones and proteases that supervise protein folding and degradation are important factors for deciding the fate of proteins whose folding is impaired by missense variations. We have studied the role of Lon and ClpXP proteases in handling of wild-type and a folding-impaired disease-associated variant (R28C) of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). Using an Escherichia coli model system, we co-overexpressed the MCAD variants and the respective proteases at two conditions: at 31 degrees C where R28C MCAD protein folds partially and at 37 degrees C where it misfolds and aggregates. Co-overexpression of Lon protease considerably accelerated the degradation rate of a pool of R28C variant MCAD synthesised during a 30min pulse and counteracted accumulation of aggregates at 37 degrees C, whereas increasing the amounts of ClpXP protease had no clear effect. Co-overexpression of either Lon or ClpXP protease markedly decreased the steady state levels of both wild-type and R28C mutant MCAD at 37 degrees C but not at 31 degrees C. Our results suggest that Lon is more efficient than ClpXP in elimination of non-native MCAD protein conformations, and accordingly, that Lon can recognise a broader spectrum of MCAD protein conformations.     

Cooperation of molecular chaperones with the ubiquitin/proteasome system

Molecular chaperones and energy-dependent proteases have long been viewed as opposing forces that control protein biogenesis. Molecular chaperones are specialized in protein folding, whereas energy-dependent proteases such as the proteasome mediate efficient protein degradation. Recent data, however, suggest that molecular chaperones directly cooperate with the ubiquitin/proteasome system during protein quality control in eukaryotic cells. Modulating the intracellular balance of protein folding and protein degradation may open new strategies for the treatment of human diseases that involve chaperone pathways such as cancer and diverse amyloid diseases.