Chaperone function of FkpA, a heat shock prolyl isomerase, in the periplasm of Escherichia coli

The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31. This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein. In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm.     

The sigmaE and Cpx regulatory pathways: overlapping but distinct envelope stress responses.

The Cpx and sigmaE extracytoplasmic stress responses sense and respond to misfolded proteins in the bacterial envelope. Recent studies have highlighted differences between these regulatory pathways in terms of activating signals, mechanisms of signal transduction and the nature of the responses. Cumulatively, the findings suggest distinct physiological roles for these partially overlapping envelope stress responses. The sigmaE pathway is essential for survival and is primarily responsible for monitoring and responding to alterations in outer membrane protein folding. Mounting evidence suggests that the Cpx regulon may have been adapted to ensure properly timed expression and assembly of adhesive organelles.     

Anti-proliferative effects of heating on the human prostatic carcinoma cells in culture

Prostatic cancers are well-known to be sensitive to heat stress. However, the mechanism by which the cancer cells are killed by high temperature remains poorly understood. The present study was undertaken to determine the anti-proliferative effects of heat stress on the prostatic cancer cells in culture. Heat shock at 43 degrees C inhibited the cell growth of three different prostatic cell lines. Flow cytometrical analysis using BrdU and PI showed a decrease in the proportion of cells in an S phase, accompanied by cell accumulation in G1 and G2, in both JCA-1 and PC-3 but not in LNcap. Both JCA-1 and PC-3 presented a strong expression of hsp70 at 37 degrees C. The heat shock caused apparent enhancement of the expression of hsp70 through the cell cycle. A treatment at 43 degrees C for 8 hours resulted in not only an apparent increment of positive hsp70 cells, but cells with subdiploid DNA content in LNcap. Flow cytometrical analysis by FITC-labeled Annexin V showed increment of apoptotic cells at 43 degrees C for 8 hours in LNcap cells. The results suggest that apoptosis is an important pathway of heat-induced killing of these cells. In conclusion, the cell growth of prostatic cancers may be affected by the temperature through relationship of the cell cycle and hsp70.     

Cycloheximide can rescue heat-shocked L cells from death by blocking stress-induced apoptosis.

Cultured mouse L cells undergo apoptosis upon 1 h heat shock at 43 and 45 degrees C. Morphologically characteristic apoptotic cells begin to appear soon after the shock. Immunohistochemistry with anti-transglutaminase antibody shows that in most treated cells the enzyme is induced. Its activation results in the formation of highly cross-linked detergent-resistant apoptotic bodies during recovery. Cycloheximide added during hyperthermic stress inhibits the appearance of apoptotic bodies, showing that heat-shock-induced apoptosis is dependent on protein neosynthesis. The analysis of colony-forming ability of heat-shocked L cells shows a survival of 5% at 43 degrees C and less than 0.02% at 45 degrees C. When protein synthesis is inhibited during heat shock the fraction of surviving cells increases to 23% at 43 degrees C and 0.9% at 45 degrees C. This suggest that part of the cells that die upon heat shock are not heavily damaged and would have survived in the presence of a block in protein synthesis.     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

Absence of the outer membrane phospholipase A suppresses the temperature-sensitive phenotype of Escherichia coli degP mutants and induces the Cpx and sigma(E) extracytoplasmic stress responses

DegP is a periplasmic protease that is a member of both the sigma(E) and Cpx extracytoplasmic stress regulons of Escherichia coli and is essential for viability at temperatures above 42 degrees C. [U-(14)C]acetate labeling experiments demonstrated that phospholipids were degraded in degP mutants at elevated temperatures. In addition, chloramphenicol acetyltransferase, beta-lactamase, and beta-galactosidase assays as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that large amounts of cellular proteins are released from degP cells at the nonpermissive temperature. A mutation in pldA, which encodes outer membrane phospholipase A (OMPLA), was found to rescue degP cells from the temperature-sensitive phenotype. pldA degP mutants had a normal plating efficiency at 42 degrees C, displayed increased viability at 44 degrees C, showed no degradation of phospholipids, and released far lower amounts of cellular protein to culture supernatants. degP and pldA degP mutants containing chromosomal lacZ fusions to Cpx and sigma(E) regulon promoters indicated that both regulons were activated in the pldA mutants. The overexpression of the envelope lipoprotein, NlpE, which induces the Cpx regulon, was also found to suppress the temperature-sensitive phenotype of degP mutants but did not prevent the degradation of phospholipids. These results suggest that the absence of OMPLA corrects the degP temperature-sensitive phenotype by inducing the Cpx and sigma(E) regulons rather than by inactivating the phospholipase per se.     

Heat Shock Response Activation Exacerbates Inclusion Body Formation in a Cellular Model of Huntington Disease

The cellular heat shock response (HSR) protects cells from toxicity associated with defective protein folding, and this pathway is widely viewed as a potential pharmacological target to treat neurodegenerative diseases linked to protein aggregation. Here we show that the HSR is not activated by mutant huntingtin (HTT) even in cells selected for the highest expression levels and for the presence of inclusion bodies containing aggregated protein. Surprisingly, HSR activation by HSF1 overexpression or by administration of a small molecule activator lowers the concentration threshold at which HTT forms inclusion bodies in cells expressing aggregation-prone, polyglutamine-expanded fragments of HTT. These data suggest that the HSR does not mitigate inclusion body formation.     

A third envelope stress signal transduction pathway in Escherichia coli

Escherichia coli uses overlapping envelope stress responses to adapt to insults to the bacterial envelope that cause protein misfolding. The sigmaE and Cpx envelope stress responses are activated by both common and distinct envelope stresses and respond by increasing the expression of the periplasmic protease DegP as well as target genes unique to each response. The sigmaE pathway is involved in outer membrane protein (OMP) folding quality control whereas the Cpx pathway plays an important role in the assembly of at least one pilus. Previously, we identified the spy gene as a new Cpx regulon member of unknown function. Interestingly, induction of spy expression by severe envelope stresses such as spheroplasting is only partially dependent on an intact Cpx signalling pathway, unlike other Cpx-regulated genes. Here we show that the BaeS sensor kinase and BaeR response regulator also control expression of spy in response to envelope stress. BaeS and BaeR do not affect expression of other known Cpx-regulated genes, however, baeR cpxR double mutants show increased sensitivity to envelope stresses relative to either single mutant alone. We propose that the Bae signal transduction pathway controls a third envelope stress response in E. coli that induces expression of a distinct set of adaptive genes.     

Identification of inducers of the Yersinia enterocolitica phage shock protein system and comparison to the regulation of the RpoE and Cpx extracytoplasmic stress responses

Known inducers of the phage shock protein (Psp) system suggest that it is an extracytoplasmic stress response, as are the well-studied RpoE and Cpx systems. However, a random approach to identify conditions and proteins that induce the Psp system has not been attempted. It is also unknown whether the proteins or mutations that induce Psp are specific or if they also activate the RpoE and Cpx systems. This study addressed these issues for the Yersinia enterocolitica Psp system. Random transposon mutagenesis identified null mutations and overexpression mutations that increase Phi(pspA-lacZ) operon fusion expression. The results suggest that Psp may respond exclusively to extracytoplasmic stress. Null mutations affected glucosamine-6-phosphate synthetase (glmS), which plays a role in cell envelope biosynthesis, and the F0F1 ATPase (atp operon). The screen also revealed that in addition to several secretins, the overexpression of three novel putative inner membrane proteins (IMPs) induced the Psp response. We also compared induction of the Y. enterocolitica Psp, RpoE, and Cpx responses. Overexpression of secretins or the three IMPs or the presence of an atpB null mutation only induced the Psp response. Similarly, known inducers of the RpoE and Cpx responses did not significantly induce the Psp response. Only the glmS null mutation induced all three responses. Therefore, Psp is induced distinctly from the RpoE and Cpx systems. The specific IMP inducers may be valuable tools to probe specific signal transduction events of the Psp response in future studies.     

RseB binding to the periplasmic domain of RseA modulates the RseA:sigmaE interaction in the cytoplasm and the availability of sigmaE.RNA polymerase.

The Escherichia coli sigmaE regulon has evolved to sense the presence of misfolded proteins in the bacterial envelope. Expression of periplasmic chaperones and folding catalysts is under the control of sigmaE RNA polymerase. The N-terminal domain of RseA sequesters sigmaE in the cytoplasmic membrane, preventing its association with core RNA polymerase. The C-terminal domain of RseA interacts with RseB, a periplasmic protein. The relative concentration of sigmaE:RseA:RseB is 2:5:1 and this ratio remains unaltered upon heat shock induction of the sigmaE regulon. Purification from crude cellular extracts yields cytoplasmic, soluble sigmaE RNA polymerase as well as membrane sequestered sigmaE.RseA and sigmaE.RseA.RseB. RseB binding to the C-terminal domain of RseA increases the affinity of RseA for sigmaE by 2- to 3-fold (Kd 50-100 nM). RseB binds also to the misfolded aggregates of MalE31, a variant of maltose binding protein that forms inclusion bodies in the periplasm. We discuss a model whereby the RseB-RiseA interaction represents a measure for misfolded polypeptides in the bacterial envelope, modulating the assembly of sigmaE RNA polymerase and the cellular heat shock response.     

Parameters for cellular viability and membrane function in chenopodium cells show a specific response of extracellular pH to heat shock with extreme Q10

The effect of brief heat shock on Chenopodium cells was investigated by measuring biochemical parameters for cellular vitality, membrane function and integrity: extracellular pH, release of osmotic compounds, phosphatase, protein and betalain, and cellular reduction of DCPIP and MTT. A threshold temperature was found at 45 degrees C, where release of osmotic compounds, protein and betalain, and reduction of DCPIP and MTT indicate loss of vitality. Extracellular pH and an alkaline phosphatase responded 10-20 degrees C below this threshold, suggesting that extracellular alkalinization, and probably the release of a phosphatase, are part of a specific cellular response to abiotic stress induced by heat shock. The extracellular proton concentration did not increase above 45 degrees C: this may indicate equilibration of gradients driving this process or an inactivation of cellular mechanisms responsible for extracellular alkalinization. The response of extracellular pH to heat shock in Chenopodium cell suspensions was fast, i.e., up to +1 pH in 5 min. Addition of the K+/H+ antiporter nigericin to Chenopodium cells caused an extracellular alkalinization similar to heat shock. The heat shock-induced extracellular alkalinization was characterized by Q10 values for distinct ranges of temperature (Q10 of 56 for 24-31 degrees C, 2.3 for 31-42 degrees C, and 1.0 for 42-50 degrees C). To the author's knowledge, the Q10 of 56 is the highest found up to now. These results suggest that extracellular protons are involved in temperature sensing and signalling in plant cells, probably via a channel-mediated pathway.     

Tethering of CpxP to the inner membrane prevents spheroplast induction of the cpx envelope stress response

The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP-CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the sigmaE stress response. Because the sigmaE response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process.     

Crystal structure of a defective folding protein

Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.     

Characterization of goldfish heat shock protein-30 induced upon severe heat shock in cultured cells

Temperature-dependent changes of growth rate and protein components were investigated for primary cultured cells derived from goldfish caudal fin. When the culture temperature was shifted from 20 degrees C to 35 degrees C and 40 degrees C, the growth rate was increased at 35 degrees C as compared with that at 20 degrees C, but no cell growth was observed at 40 degrees C. The differential scanning calorimetry demonstrated the onset of the endothermic reaction for goldfish cellular components at 40 degrees C. Therefore, the temperature shift to 40 degrees C was found to be of severe heat shock for goldfish cultured cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that, although expression of 70-kDa components was slightly induced at 35 degrees C, the temperature shift to 40 degrees C markedly induced the expression of the 30-kDa component in addition to that of 70-kDa component. The N-terminal amino acid sequencing identified the 30- and 70-kDa components to be heat shock protein (Hsp)-30 and Hsp70, respectively. Northern blot analysis revealed that the enhanced Hsp30 messenger ribonucleic acid (mRNA) levels were only observed at 40 degrees C, whereas Hsp70 mRNA was slightly accumulated at 35 degrees C. These results indicated that Hsp30 might have important functions under severe heat stress condition.     

Bacterial inclusion bodies are cytotoxic in vivo in absence of functional chaperones DnaK or GroEL

Cytotoxicity of cytoplasmic bacterial inclusion bodies has been explored in vivo in cells producing a model, misfolding-prone beta-galactosidase fusion protein. The formation of such aggregates does not result in detectable toxicity on Escherichia coli producing cells. However, a deficiency in the main chaperones DnaK or GroEL but not in other components of the heat shock system such as the chaperone ClpA or the protease Lon, promotes a dramatic inhibition of cell growth. The role of DnaK and GroEL in minimizing toxicity of in vivo protein aggregation is discussed in the context of the conformational stress and the protein quality control system.