scholarly journals Feeling the Heat: The Campylobacter jejuni HrcA Transcriptional Repressor Is an Intrinsic Protein Thermosensor

Biomolecules ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 1413
Author(s):  
Giovanni Versace ◽  
Marta Palombo ◽  
Anna Menon ◽  
Vincenzo Scarlato ◽  
Davide Roncarati
Keyword(s):  
Heat Stress ◽  
Heat Shock ◽  
Dna Binding ◽  
Campylobacter Jejuni ◽  
Heat Shock Response ◽  
Binding Activity ◽  
Protective Mechanism ◽  
Intrinsic Protein ◽  
Dna Binding Activity ◽  
Shock Response

The heat-shock response, a universal protective mechanism consisting of a transcriptional reprogramming of the cellular transcriptome, results in the accumulation of proteins which counteract the deleterious effects of heat-stress on cellular polypeptides. To quickly respond to thermal stress and trigger the heat-shock response, bacteria rely on different mechanisms to detect temperature variations, which can involve nearly all classes of biological molecules. In Campylobacter jejuni the response to heat-shock is transcriptionally controlled by a regulatory circuit involving two repressors, HspR and HrcA. In the present work we show that the heat-shock repressor HrcA acts as an intrinsic protein thermometer. We report that a temperature upshift up to 42°C negatively affects HrcA DNA-binding activity to a target promoter, a condition required for de-repression of regulated genes. Furthermore, we show that this impairment of HrcA binding at 42°C is irreversible in vitro, as DNA-binding was still not restored by reversing the incubation temperature to 37°C. On the other hand, we demonstrate that the DNA-binding activity of HspR, which controls, in combination with HrcA, the transcription of chaperones’ genes, is unaffected by heat-stress up to 45°C, portraying this master repressor as a rather stable protein. Additionally, we show that HrcA binding activity is enhanced by the chaperonin GroE, upon direct protein–protein interaction. In conclusion, the results presented in this work establish HrcA as a novel example of intrinsic heat-sensing transcriptional regulator, whose DNA-binding activity is positively modulated by the GroE chaperonin.

scholarly journals Cooperative Regulation of Campylobacter jejuni Heat-Shock Genes by HspR and HrcA

2020 ◽  
Vol 8 (8) ◽  
pp. 1161
Author(s):  
Marta Palombo ◽  
Vincenzo Scarlato ◽  
Davide Roncarati
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Campylobacter Jejuni ◽  
Heat Shock Response ◽  
Inverted Repeat ◽  
Regulatory Function ◽  
Heat Shock Genes ◽  
Repressive Effect ◽  
Shock Response ◽  
Regulated Promoters

The heat-shock response is defined by the transient gene-expression program that leads to the rapid accumulation of heat-shock proteins. This evolutionary conserved response aims at the preservation of the intracellular environment and represents a crucial pathway during the establishment of host–pathogen interaction. In the food-borne pathogen Campylobacter jejuni two transcriptional repressors, named HspR and HrcA, are involved in the regulation of the major heat-shock genes. However, the molecular mechanism underpinning HspR and HrcA regulatory function has not been defined yet. In the present work, we assayed and mapped the HspR and HrcA interactions on heat-shock promoters by high-resolution DNase I footprintings, defining their regulatory circuit, which governs C. jejuni heat-shock response. We found that, while DNA-binding of HrcA covers a compact region enclosing a single inverted repeat similar to the so-called Controlling Inverted Repeat of Chaperone Expression (CIRCE) sequence, HspR interacts with multiple high- and low-affinity binding sites, which contain HspR Associated Inverted Repeat (HAIR)-like sequences. We also explored the DNA-binding properties of the two repressors competitively on their common targets and observed, for the first time, that HrcA and HspR can directly interact and their binding on co-regulated promoters occurs in a cooperative manner. This mutual cooperative mechanism of DNA binding could explain the synergic repressive effect of HspR and HrcA observed in vivo on co-regulated promoters. Peculiarities of the molecular mechanisms exerted by HspR and HrcA in C. jejuni are compared to the closely related bacterium H. pylori that uses homologues of the two regulators.

scholarly journals Cytoplasmic protein misfolding titrates Hsp70 to activate nuclear Hsf1

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Anna E Masser ◽  
Wenjing Kang ◽  
Joydeep Roy ◽  
Jayasankar Mohanakrishnan Kaimal ◽  
Jany Quintana-Cordero ◽  
...  
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Protein Misfolding ◽  
Regulatory System ◽  
Binding Activity ◽  
Cytoplasmic Protein ◽  
Dna Binding Activity ◽  
Shock Response ◽  
Transcriptional Output ◽  
Substrate Binding Domain

Hsf1 is an ancient transcription factor that responds to protein folding stress by inducing the heat-shock response (HSR) that restore perturbed proteostasis. Hsp70 chaperones negatively regulate the activity of Hsf1 via stress-responsive mechanisms that are poorly understood. Here, we have reconstituted budding yeast Hsf1-Hsp70 activation complexes and find that surplus Hsp70 inhibits Hsf1 DNA-binding activity. Hsp70 binds Hsf1 via its canonical substrate binding domain and Hsp70 regulates Hsf1 DNA-binding activity. During heat shock, Hsp70 is out-titrated by misfolded proteins derived from ongoing translation in the cytosol. Pushing the boundaries of the regulatory system unveils a genetic hyperstress program that is triggered by proteostasis collapse and involves an enlarged Hsf1 regulon. The findings demonstrate how an apparently simple chaperone-titration mechanism produces diversified transcriptional output in response to distinct stress loads.

Heat shock protects L6 myotubes from catabolic effects of dexamethasone and prevents downregulation of NF-κB

2001 ◽  
Vol 281 (4) ◽  
pp. R1193-R1200 ◽  
Author(s):  
Guangju Luo ◽  
Xiaoyan Sun ◽  
Eric Hungness ◽  
Per-Olof Hasselgren
Keyword(s):  
Heat Shock ◽  
Protein Degradation ◽  
Heat Shock Response ◽  
Muscle Cells ◽  
Binding Activity ◽  
Mrna Levels ◽  
Dna Binding Activity ◽  
Heat Shock Stress ◽  
L6 Myotubes ◽  
Shock Response

Glucocorticoids are the most important mediator of muscle cachexia in various catabolic conditions. Recent studies suggest that the transcription factor NF-κB acts as a suppressor of genes in the ubiquitin-proteasome proteolytic pathway and that glucocorticoids increase muscle proteolysis by downregulating NF-κB activity. The heat shock (stress) response, characterized by the induction of heat shock proteins, confers a protective effect against a variety of harmful stimuli. In the present study, we tested the hypothesis that the heat shock response protects muscle cells from the catabolic effects of dexamethasone and prevents downregulation of NF-κB. Cultured L6 myotubes were subjected to heat shock (43°C for 1 h) followed by recovery at 37°C for 1 h. Thereafter, cells were treated for 6 h with 1 μM dexamethasone, during which period protein degradation was measured as release of TCA-soluble radioactivity from proteins that had been prelabeled with [3H]tyrosine. Heat shock resulted in increased protein and mRNA levels for heat shock protein 70. The increase in protein degradation induced by dexamethasone was prevented in cells expressing the heat shock response. In the same cells, dexamethasone-induced downregulation of NF-κB DNA binding activity was blocked. The present results suggest that the heat shock response may protect muscle cells from the catabolic effects of dexamethasone and that this effect of heat shock may be related to inhibited downregulation of NF-κB activity.

Developmentally regulated nuclear transport of transcription factors in Drosophila embryos enable the heat shock response

Development ◽  
1998 ◽  
Vol 125 (23) ◽  
pp. 4841-4850 ◽  
Author(s):  
Z. Wang ◽  
S. Lindquist
Keyword(s):  
Transcription Factors ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Nuclear Transport ◽  
Binding Activity ◽  
Spatiotemporal Pattern ◽  
Dna Binding Activity ◽  
Early Embryos ◽  
Shock Response ◽  
Drosophila Embryos

Hsp70 is a broadly conserved thermotolerance factor, but inhibits growth at normal temperatures and cannot be induced in early embryos. We report that in Drosophila embryos the temporal and spatial patterns of Hsp70 inducibility were unexpectedly complex, with striking differences between the soma and the germline. In both, regulation occurred at the level of transcription. During the refractory period for Hsp70 induction, HSF (heat-shock transcription factor) exhibited specific DNA-binding activity characteristic of activation in extracts of heated embryos. Remarkably, however, HSF was restricted to the cytoplasm in intact embryos even after heat shock. HSF moved from the cytoplasm to the nucleus in the absence of heat precisely when the capacity to induce Hsp70 was acquired (cycle 12 of the germline, cycle 13 in the soma). During oogenesis, Hsp70 inducibility was lost in nurse cells around stage 10, in a posterior-to-anterior gradient and HSF redistributed from nucleus to cytoplasm in the same spatiotemporal pattern. In a highly inbred derivative of the Samarkind strain, HSF moved into embryonic nuclei earlier than in our standard wild-type strain. Correspondingly, Hsp70 was inducible earlier, confirming that nuclear transport of HSF controls the inducibility of Hsp70 in early embryos. We also report for the first time the nuclear import patterns of two general transcription factors, RNA polymerase subunit Ilc and TATA binding protein (TBP). Both enter nuclei in a highly synchronous manner, independently of each other and of HSF. The import of TBP coincides with the first reported appearance of transcripts in the embryo. We suggest that the potentiation of general and heat shock-specific transcription in Drosophila embryos is controlled by the developmentally programmed relocalization of general and heat shock-specific transcription factors. Restricted nuclear entry of HSF represents a newly described mechanism for regulating the heat-shock response.

Interaction between heat shock factor and hsp70 is insufficient to suppress induction of DNA-binding activity in vivo

1994 ◽  
Vol 14 (10) ◽  
pp. 6552-6560
Author(s):  
S K Rabindran ◽  
J Wisniewski ◽  
L Li ◽  
G C Li ◽  
C Wu
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Heat Shock Proteins ◽  
Expression System ◽  
Stress Protein ◽  
Binding Activity ◽  
Protein Family ◽  
Oligomeric State ◽  
Dna Binding Activity ◽  
Shock Proteins

The intracellular level of free heat shock proteins, in particular the 70-kDa stress protein family, has been suggested to be the basis of an autoregulatory mechanism by which the cell measures the level of thermal stress and regulates the synthesis of heat shock proteins. It has been proposed that the DNA-binding and oligomeric state of the heat shock transcription factor (HSF) is a principal step in the induction pathway that is responsive to the level of 70-kDa stress protein. To test this hypothesis, we investigated the association between HSF and 70-kDa stress protein by means of a coimmunoprecipitation assay. We found that 70-kDa stress proteins associate to similar extents with both latent and active forms of HSF, although unlike other 70-kDa stress protein substrates, the association with HSF was not significantly disrupted in the presence of ATP. Gel mobility shift assays indicated that active HSF trimers purified from a bacterial expression system could not be substantially deactivated in vitro with purified 70-kDa stress protein and ATP. In addition, elevated concentrations of hsp70 alone could not significantly inhibit induction of the DNA-binding activity of endogenous HSF in cultured rat cells, and the induction was also not inhibited in cultured rat cells or Drosophila cells containing elevated levels of all members of the heat shock protein family. However, the deactivation of HSF to the non-DNA-binding state after prolonged heat stress or during recovery could be accelerated by increased levels of heat shock proteins. Hence, the level of heat shock proteins may affect the rate of disassembly of HSF trimers, but another mechanism, as yet undefined, appears to control the onset of the oligomeric transitions.

Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells

1992 ◽  
Vol 12 (9) ◽  
pp. 4104-4111
Author(s):  
L Sistonen ◽  
K D Sarge ◽  
B Phillips ◽  
K Abravaya ◽  
R I Morimoto
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Binding Activity ◽  
Erythroid Cell ◽  
Hsp70 Gene ◽  
Mobility Shift ◽  
Heat Shock Element ◽  
Dna Binding Activity ◽  
Erythroleukemia Cells

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.

scholarly journals The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein.

1990 ◽  
Vol 9 (1) ◽  
pp. 69-76 ◽  
Author(s):  
A. Wilhelmsson ◽  
S. Cuthill ◽  
M. Denis ◽  
A.C. Wikström ◽  
J.A. Gustafsson ◽  
...  
Keyword(s):  
Heat Shock ◽  
Dna Binding ◽  
Heat Shock Protein ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Dioxin Receptor

scholarly journals Molecular Basis of the Defective Heat Stress Response in Mycobacterium leprae

2007 ◽  
Vol 189 (24) ◽  
pp. 8818-8827 ◽  
Author(s):  
Diana L. Williams ◽  
Tana L. Pittman ◽  
Mike Deshotel ◽  
Sandra Oby-Robinson ◽  
Issar Smith ◽  
...  
Keyword(s):  
Heat Stress ◽  
Stress Response ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Elevated Temperatures ◽  
Transcriptional Response ◽  
Mycobacterium Leprae ◽  
Heat Stress Response ◽  
Regulatory Circuits ◽  
Shock Response

ABSTRACT Mycobacterium leprae, a major human pathogen, grows poorly at 37°C. The basis for its inability to survive at elevated temperatures was investigated. We determined that M. leprae lacks a protective heat shock response as a result of the lack of transcriptional induction of the alternative sigma factor genes sigE and sigB and the major heat shock operons, HSP70 and HSP60, even though heat shock promoters and regulatory circuits for these genes appear to be intact. M. leprae sigE was found to be capable of complementing the defective heat shock response of mycobacterial sigE knockout mutants only in the presence of a functional mycobacterial sigH, which orchestrates the mycobacterial heat shock response. Since the sigH of M. leprae is a pseudogene, these data support the conclusion that a key aspect of the defective heat shock response in M. leprae is the absence of a functional sigH. In addition, 68% of the genes induced during heat shock in M. tuberculosis were shown to be either absent from the M. leprae genome or were present as pseudogenes. Among these is the hsp/acr2 gene, whose product is essential for M. tuberculosis survival during heat shock. Taken together, these results suggest that the reduced ability of M. leprae to survive at elevated temperatures results from the lack of a functional transcriptional response to heat shock and the absence of a full repertoire of heat stress response genes, including sigH.

Sub-lethal heat stress causes apoptosis in an Antarctic fish that lacks an inducible heat shock response

2014 ◽  
Vol 44 ◽  
pp. 119-125 ◽  
Author(s):  
Isaac M. Sleadd ◽  
Marissa Lee ◽  
Daniel O. Hassumani ◽  
Tonya M.A. Stecyk ◽  
Otto K. Zeitz ◽  
...  
Keyword(s):  
Heat Stress ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Antarctic Fish ◽  
Shock Response

scholarly journals Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (1) ◽  
pp. 281-288 ◽  
Author(s):  
G J Gallo ◽  
T J Schuetz ◽  
R E Kingston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Dna Binding ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Heat Shock Response ◽  
Heat Shock Factor ◽  
Regulatory Sequence ◽  
Heat Shock Element ◽  
Shock Response

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes. We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe. HSF binding was not observed in extracts from normally growing S. pombe (28 degrees C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45 degrees C. This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures. The S. pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis. The induction of S. pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated. S. pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa. Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S. pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.

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