Cloning and characterization of two cellulase genes from Lentinula edodes

The effect of overproduction of the Hsp70 system proteins (DnaK, DnaJ, GrpE) and/or ClpB (Hsp100) from plasmids on the process of formation and removal of heat-aggregated proteins from Escherichia coli cells (the S fraction) was investigated by sucrose density gradient centrifugation. Two plasmids were employed: pKJE7 carrying the dnaK/dnaJ/grpE genes under the control of the araB promoter and pClpB carrying the clpB gene under the control of its own promoter (sigma(32)-dependent). In the wild-type cells the S fraction after 15 min of heat shock amounted to 21% of cellular insoluble proteins (IP), and disappeared 10 min after transfer of the culture to 37 degrees C. In contrast to this, in the clpB mutant the S fraction was larger (35% IP) and its elimination was retarded, nearly 60% of the aggregated proteins remained stable 30 min after heat shock. This result points to the importance of ClpB in removal of the heat-aggregated proteins from cells. Overproduction of the Hsp70 system proteins (exceeding by about 1.5-fold that of wild-type) in wild-type and DeltaclpB cells completely prevented the formation of the S fraction during heat shock. Overproduction of ClpB (exceeding by about eight-fold that of wild-type) in the same background did not prevent protein aggregation after heat shock and only partly compensated for the effect of the mutation in the clpB gene. Monitoring the S fraction during co-production of DnaK/DnaJ/GrpE and ClpB in the DeltaclpB mutant revealed that both the levels of expression and the ratios of ClpB to Hsp70 system proteins had a significant effect on the formation and removal of protein aggregates in heat-shocked E. coli cells. In the presence of excess ClpB, an increase in the levels of DnaK, DnaJ and GrpE was required to prevent aggregate formation upon heat shock or to efficiently remove protein aggregates after heat shock. Therefore, it is supposed that a high level of ClpB under some conditions, especially at insufficient levels of Hsp70 system proteins, may support protein aggregation resulting from heat shock and may lead to stabilization of hydrophobic aggregates.     

Small heat shock proteins, ClpB and the DnaK system form a functional triade in reversing protein aggregation

Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co-operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co-operatively the DnaK system in reversing protein aggregation. A delta ibpAB delta clpB double mutant exhibits strongly increased protein aggregation at 42 degrees C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37 degrees C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for delta ibpAB, delta clpB, and the double delta ibpAB delta clpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.     

The Porphyromonas gingivalis clpB gene is involved in cellular invasion in vitro and virulence in vivo

ClpB, a component of stress response in microorganisms, serves as a chaperone, preventing protein aggregation and assisting in the refolding of denatured proteins. A clpB mutant of Porphyromonas gingivalis W83 demonstrated increased sensitivity to heat stress, but not to hydrogen peroxide and extreme pHs. In KB cells, human coronary artery endothelial (HCAE) cells and gingival epithelial cells, the clpB mutant exhibited significantly decreased invasion suggesting that the ClpB protein is involved in cellular invasion. Transmission electron microscopic analysis showed that the clpB mutant was more susceptible to intracellular killing than the wild-type strain in HCAE cells. The global genetic profile of the clpB mutant showed that 136 genes belonging to several different cellular function groups were differentially regulated, suggesting that ClpB is ultimately involved in the expression of multiple P. gingivalis genes. A competition assay in which a mixture of wild-type W83 and the clpB mutant were injected into mice demonstrated that the clpB mutant did not survive as well as the wild type. Additionally, mice treated with the clpB mutant alone survived significantly better than those treated with the wild-type strain. Collectively, these data suggest that ClpB, either directly or indirectly, plays an important role in P. gingivalis virulence.     

The Escherichia coli heat shock protein ClpB restores acquired thermotolerance to a cyanobacterial clpB deletion mutant

In both prokaryotes and eukaryotes, the heat shock protein ClpB functions as a molecular chaperone and plays a key role in resisting high temperature stress. ClpB is important for the development of thermotolerance in yeast and cyanobacteria but apparently not in Escherichia coli. We undertook a complementation study to investigate whether the ClpB protein from E coli (EcClpB) differs functionally from its cyanobacterial counterpart in the unicellular cyanobacterium Synechococcus sp. PCC 7942. The EcClpB protein is 56% identical to its ClpB1 homologue in Synechococcus. A plasmid construct was prepared containing the entire E coli clpB gene under the control of the Synechococcus clpB1 promoter. This construct was transformed into a Synechococcus clpB1 deletion strain (deltaclpB1) and integrated into a phenotypically neutral site of the chromosome. The full-length EcClpB protein (EcClpB-93) was induced in the transformed Synechococcus strain during heat shock as well as the smaller protein (EcClpB-79) that arises from a second translational start inside the single clpB message. Using cell survival measurements we show that the EcClpB protein can complement the Synechococcus deltaclpB1 mutant and restore its ability to develop thermotolerance. We also demonstrate that both EcClpB-93 and -79 appear to contribute to the degree of acquired thermotolerance restored to the Synechococcus complementation strains.     

The importance of having thermosensor control in the DnaK chaperone system

In addition to the sigma(32)-mediated heat shock response, the DnaK/DnaJ/GrpE molecular chaperone system of Escherichia coli directly adapts to elevated temperatures by sequestering a higher fraction of substrate. This immediate heat shock response is due to the differential temperature dependence of the activity of DnaJ, which stimulates the hydrolysis of DnaK-bound ATP, and the activity of GrpE, which facilitates ADP/ATP exchange and converts DnaK from its high-affinity ADP-liganded state into its low-affinity ATP-liganded state. GrpE acts as thermosensor with its ADP/ATP exchange activity decreasing above 40 degrees C. To assess the importance of this reversible thermal adaptation for the chaperone action of the DnaK/DnaJ/GrpE system during heat shock, we used glucose-6-phosphate dehydrogenase and luciferase as substrates. We compared the performance of wild-type GrpE as a component of the chaperone system with that of GrpE R40C. In this mutant, the thermosensing helices are stabilized with an intersubunit disulfide bond and its nucleotide exchange activity thus increases continuously with increasing temperature. Wild-type GrpE with intact thermosensor proved superior to GrpE R40C with desensitized thermosensor. The chaperone system with wild-type GrpE yielded not only a higher fraction of refolding-competent protein at the end of a heat shock but also protected luciferase more efficiently against inactivation during heat shock. Consistent with their differential thermal behavior, the protective effects of wild-type GrpE and GrpE R40C diverged more and more with increasing temperature. Thus, the direct thermal adaptation of the DnaK chaperone system by thermosensing GrpE is essential for efficient chaperone action during heat shock.     

Reduced adipose tissue mass and hypoleptinemia in iNOS deficient mice: effect of LPS on plasma leptin and adiponectin concentrations

The AAA+ chaperone ClpB solubilizes in cooperation with the DnaK chaperone system aggregated proteins. The mechanistic features of the protein disaggregation process are poorly understood. Here, we investigated the mechanism of ClpB/DnaK-dependent solubilization of heat-aggregated malate dehydrogenase (MDH) by following characteristics of MDH aggregates during the disaggregation reaction. We demonstrate that disaggregation is achieved by the continuous extraction of unfolded MDH molecules and not by fragmentation of large MDH aggregates. These findings support a ClpB-dependent threading mechanism as an integral part of the disaggregation reaction.     

M domains couple the ClpB threading motor with the DnaK chaperone activity

The AAA(+) chaperone ClpB mediates the reactivation of aggregated proteins in cooperation with the DnaK chaperone system. ClpB consists of two AAA domains that drive the ATP-dependent threading of substrates through a central translocation channel. Its unique middle (M) domain forms a coiled-coil structure that laterally protrudes from the ClpB ring and is essential for aggregate solubilization. Here, we demonstrate that the conserved helix 3 of the M domain is specifically required for the DnaK-dependent shuffling of aggregated proteins, but not of soluble denatured substrates, to the pore entrance of the ClpB translocation channel. Helix 3 exhibits nucleotide-driven conformational changes possibly involving a transition between folded and unfolded states. This molecular switch controls the ClpB ATPase cycle by contacting the first ATPase domain and establishes the M domain as a regulatory device that acts in the disaggregation process by coupling the threading motor of ClpB with the DnaK chaperone activity.     

Cooperation between two ClpB isoforms enhances the recovery of the recombinant β-galactosidase from inclusion bodies

Bacterial ClpB is a molecular chaperone that solubilizes and reactivates aggregated proteins in cooperation with the DnaK chaperone system. The mechanism of protein disaggregation mediated by ClpB is linked to translocation of substrates through the central channel within the ring-hexameric structure of ClpB. Two isoforms of ClpB are produced in vivo: the full-length ClpB95 and the truncated ClpB80 (ClpBΔN), which does not contain the N-terminal domain. The functional specificity of the two ClpB isoforms and the biological role of the N-terminal domain are still not fully understood. Recently, it has been demonstrated that ClpB may achieve its full potential as an aggregate-reactivating chaperone through the functional interaction and synergistic cooperation of its two isoforms. It has been found that the most efficient resolubilization and reactivation of stress-aggregated proteins occurred in the presence of both ClpB95 and ClpB80. In this work, we asked if the two ClpB isoforms functionally cooperate in the solubilization and reactivation of proteins from insoluble inclusion bodies (IBs) in Escherichia coli cells. Using the model β-galactosidase fusion protein (VP1LAC), we found that solubilization and reactivation of enzymes entrapped in IBs occurred more efficiently in the presence of ClpB95 with ClpB80 than with either ClpB95 or ClpB80 alone. The two isoforms of ClpB chaperone acting together enhanced the solubility and enzymatic activity of β-galactosidase sequestered into IBs. Both ClpB isoforms were associated with IBs of β-galactosidase, what demonstrates their affinity to this type of aggregates. These results demonstrate a synergistic cooperation between the two isoforms of ClpB chaperone. In addition, no significant recovery of the β-galactosidase from IBs in ΔclpB mutant cells suggests that ClpB is a key chaperone in IB protein release.     

The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp. strain PCC 7942.

The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis. A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp. strain PCC 7942. The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%). Inactivation of clpB in Synechococcus sp. strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions. In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa. Both proteins were absent in the delta clpB strain. The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged. In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity. Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type. Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp. to determine the importance of ClpB for acquired thermotolerance. Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min. By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min. Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance. These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.