Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone,... more
Genes encoding ribosomal proteins and other components of the translational apparatus are coregulated to efficiently adjust the protein synthetic capacity of the cell. Ssb, a Saccharomyces cerevisiae Hsp70 cytosolic molecular chaperone, is associated with the ribosome-nascent chain complex. To determine whether this chaperone is coregulated with ribosomal proteins, we studied the mRNA regulation of SSB under several environmental conditions. Ssb and the ribosomal protein rpL5 mRNAs were up-regulated upon carbon upshift and down-regulated upon amino acid limitation, unlike the mRNA of another cytosolic Hsp70, Ssa. Ribosomal protein and Ssb mRNAs, like many mRNAs, are down-regulated upon a rapid temperature upshift. The mRNA reduction of several ribosomal protein genes and Ssb was delayed by the presence of an allele, EXA3-1 , of the gene encoding the heat shock factor (HSF). However, upon a heat shock the EXA3-1 mutation did not significantly alter the reduction in the mRNA levels of...
The simultaneous exposure of WI‐38 human fibroblasts (HF) to a heat shock (45°C, 30 min) and an alkaline shift (≥ pH 8.0) in the incubation medium increased and extended the expression of heat shock proteins (hsps). Hsp70 was the most... more
The simultaneous exposure of WI‐38 human fibroblasts (HF) to a heat shock (45°C, 30 min) and an alkaline shift (≥ pH 8.0) in the incubation medium increased and extended the expression of heat shock proteins (hsps). Hsp70 was the most prominent inducible hsp synthesized during the recovery phase after the double shock, and the increase in synthesis depended on the degree of alkalinization during the heat shock. The accumulation of inducible hsp70, which was shown by Western blotting to occur in the late part of the recovery period, was more pronounced in the cells exposed to alkaline medium during the heat shock. Northern blotting did not reveal any increase in hsp70 mRNA, although time course studies following the double shock indicated a more prolonged presence of mRNA. Hsp70 gene activation was evaluated by a gel retardation assay using a 32P‐labelled DNA oligonucleotide containing the heat shock consensus element (HSE) and a heat shock‐induced specific binding protein (heat shoc...
Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme activated in response to DNA strand breaks, has been implicated in cell dysfunction in myocardial reperfusion injury. PARP-1 has also been shown to participate in transcription and... more
Poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme activated in response to DNA strand breaks, has been implicated in cell dysfunction in myocardial reperfusion injury. PARP-1 has also been shown to participate in transcription and regulation of gene expression. In this study, we investigated the role of PARP-1 on the signal transduction pathway of activator protein-1 (AP-1) and heat shock factor-1 (HSF-1) in myocardial reperfusion injury. Mice genetically deficient of PARP-1 (PARP-1–/–mice) exhibited a significant reduction of myocardial damage after occlusion and reperfusion of the left anterior descending branch of the coronary artery compared with their wild-type littermates. This cardioprotection was associated with a reduction of the phosphorylative activity of JNK and, subsequently, reduction of the DNA binding of the signal transduction factor AP-1. On the contrary, in PARP-1–/–mice, DNA binding of HSF-1 was enhanced and was associated with a significant increase of th...
The existence of multiple heat shock factor (HSF) genes in higher eukaryotes has promoted questions regarding the functions of these HSF family members, especially with respect to the stress response. To address these questions, we have... more
The existence of multiple heat shock factor (HSF) genes in higher eukaryotes has promoted questions regarding the functions of these HSF family members, especially with respect to the stress response. To address these questions, we have used polyclonal antisera raised against mouse HSF1 and HSF2 to examine the biochemical, physical, and functional properties of these two factors in unstressed and heat-shocked mouse and human cells. We have identified HSF1 as the mediator of stress-induced heat shock gene transcription. HSF1 displays stress-induced DNA-binding activity, oligomerization, and nuclear localization, while HSF2 does not. Also, HSF1 undergoes phosphorylation in cells exposed to heat or cadmium sulfate but not in cells treated with the amino acid analog L-azetidine-2-carboxylic acid, indicating that phosphorylation of HSF1 is not essential for its activation. Interestingly, HSF1 and HSF2 overexpressed in transfected 3T3 cells both display constitutive DNA-binding activity, ...
Two members of the heat shock transcription factor (HSF) family, HSF1 and HSF2, both function as transcriptional activators of heat shock gene expression. However, the inducible DNA-binding activities of these two factors are regulated by... more
Two members of the heat shock transcription factor (HSF) family, HSF1 and HSF2, both function as transcriptional activators of heat shock gene expression. However, the inducible DNA-binding activities of these two factors are regulated by distinct pathways. HSF1 is activated by heat shock and other forms of stress, whereas HSF2 is activated during hemin-induced differentiation of human K562 erythroleukemia cells, suggesting a role for HSF2 in regulating heat shock gene expression under nonstress conditions such as differentiation and development. To understand the distinct regulatory pathways controlling HSF2 and HSF1 activities, we have examined the biochemical and physical properties of the control and activated states of HSF2 and compared these with the properties of HSF1. Our results reveal that the inactive, non-DNA-binding forms of HSF2 and HSF1 exist primarily in the cytoplasm of untreated K562 cells as a dimer and monomer, respectively. This difference in the control oligome...
Cell and tissue injury activate the inflammatory response through the action(s) of arachidonic acid and its metabolites, leading to the expression of acute-phase proteins and inflammatory cytokines. At the molecular level, little is known... more
Cell and tissue injury activate the inflammatory response through the action(s) of arachidonic acid and its metabolites, leading to the expression of acute-phase proteins and inflammatory cytokines. At the molecular level, little is known how arachidonic acid regulates the inflammatory response. As inflammation is also associated with local increase in tissue temperatures, we examined whether arachidonic acid was directly involved in the heat shock response. Extracellular exposure to arachidonic acid induced heat shock gene transcription in a dose-dependent manner via acquisition of DNA-binding activity and phosphorylation of heat shock factor 1 (HSF1). In addition, exposure of cells to low concentrations of arachidonic acid, which by themselves did not induce HSF1 DNA-binding activity, reduced the temperature threshold for HSF1 activation from elevated temperatures which are not physiologically relevant (> 42 degrees C) to temperatures which can be attained during the febrile re...
The correlation between longevity and stress resistance observed in long-lived mutant animals suggests that the ability to sense and respond to environmental challenges could be important for the regulation of life span. We therefore... more
The correlation between longevity and stress resistance observed in long-lived mutant animals suggests that the ability to sense and respond to environmental challenges could be important for the regulation of life span. We therefore examined the role of heat shock factor (HSF-1), a master transcriptional regulator of stress-inducible gene expression and protein folding homeostasis, in the regulation of longevity. Down-regulation of hsf-1 by RNA interference suppressed longevity of mutants in an insulin-like signaling (ILS) pathway that functions in the nervous system of Caenorhabditis elegans to influence aging. hsf-1 was also required for temperature-induced dauer larvae formation in an ILS mutant. Using tissue-specific expression of wild-type or dominant negative HSF-1, we demonstrated that HSF-1 acts in multiple tissues to regulate longevity. Down-regulation of individual molecular chaperones, transcriptional targets of HSF-1, also decreased longevity of long-lived mutant but no...
Transcriptional regulation of the human hsp70 gene in response to heat shock and other forms of physiological stress occurs through the activation of heat shock transcription factor (HSF). Exposure of cells to a heat shock temperature of... more
Transcriptional regulation of the human hsp70 gene in response to heat shock and other forms of physiological stress occurs through the activation of heat shock transcription factor (HSF). Exposure of cells to a heat shock temperature of 42 degrees C results in transient activation of HSF; its DNA-binding activity increases rapidly, plateaus, and attenuates, during which the intracellular levels of hsp70 increase. In an effort to understand whether HSF is regulated negatively by hsp70, we have examined whether HSF associates with hsp70. We show that activated HSF associates with hsp70 and that the interaction is detected as the levels of hsp70 increase in the cell. Addition of ATP and other hydrolyzable nucleotides results in the dissociation of hsp70 from HSF while nonhydrolyzable nucleotide analogs do not disrupt the complex. We demonstrate that exogenous recombinant wild-type hsp70 can associate with activated HSF, whereas no association is observed with an amino-terminal or a ca...
The widely conserved heat-shock response, regulated by heat shock transcription factors, is not only essential for cellular stress resistance and adult longevity, but also for proper development. However, the genetic mechanisms by which... more
The widely conserved heat-shock response, regulated by heat shock transcription factors, is not only essential for cellular stress resistance and adult longevity, but also for proper development. However, the genetic mechanisms by which heat-shock transcription factors regulate development are not well understood. InC. elegans, we conducted an unbiased genetic screen to identify mutations that could ameliorate the developmental arrest phenotype of a heat-shock factor mutant. Here we show that loss of the conserved translational activatorrsks-1/S6-Kinase, a downstream effector of TOR kinase, can rescue the developmental-arrest phenotype of hsf-1partial loss-of-function mutants. Unexpectedly, we show that the rescue is not likely caused by reduced translation, nor to activation of any of a variety of stress-protective genes and pathways. Our findings identify an as-yet unexplained regulatory relationship between the heat-shock transcription factor and the TOR pathway duringC. elegans’...
Xenopus laevis oocytes activate transcription from the Xenopus hsp70 promoter within a chromatin template in response to heat shock. Expression of exogenous Xenopus heat shock transcription factor 1 (XHSF1) causes the activation of the... more
Xenopus laevis oocytes activate transcription from the Xenopus hsp70 promoter within a chromatin template in response to heat shock. Expression of exogenous Xenopus heat shock transcription factor 1 (XHSF1) causes the activation of the wild-type hsp70 promoter within chromatin. XHSF1 activates transcription at normal growth temperatures (18 degrees C), but heat shock (34 degrees C) facilitates transcriptional activation. Titration of chromatin in vivo leads to constitutive transcription from the wild-type hsp70 promoter. The Y box elements within the hsp70 promoter facilitate transcription in the presence or absence of chromatin. The presence of the Y box elements prevents the assembly of canonical nucleosomal arrays over the promoter and facilitates transcription. In a mutant hsp70 promoter lacking Y boxes, exogenous XHSF1 activates transcription from a chromatin template much more efficiently under heat shock conditions. Activation of transcription from the mutant promoter by exog...
Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110-124 amino acids). BAG3 is the only member of the... more
Bcl2-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that interacts with the ATPase domain of the heat shock protein (Hsp) 70 through BAG domain (110-124 amino acids). BAG3 is the only member of the family to be induced by stressful stimuli, mainly through the activity of heat shock factor 1 on bag3 gene promoter. In addition to the BAG domain, BAG3 contains also a WW domain and a proline-rich (PXXP) repeat, that mediate binding to partners different from Hsp70. These multifaceted interactions underlie BAG3 ability to modulate major biological processes, that is, apoptosis, development, cytoskeleton organization and autophagy, thereby mediating cell adaptive responses to stressful stimuli. In normal cells, BAG3 is constitutively present in a very few cell types, including cardiomyocytes and skeletal muscle cells, in which the protein appears to contribute to cell resistance to mechanical stress. A growing body of evidence indicate that BAG3 is inste...
Background Plant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling... more
Background Plant Ca2+ signals are involved in a wide array of intracellular signaling pathways after pest invasion. Ca2+-binding sensory proteins such as Ca2+-dependent protein kinases (CPKs) have been predicted to mediate the signaling following Ca2+ influx after insect herbivory. However, until now this prediction was not testable. Results To investigate the roles CPKs play in a herbivore response-signaling pathway, we screened the characteristics of Arabidopsis CPK mutants damaged by a feeding generalist herbivore, Spodoptera littoralis. Following insect attack, the cpk3 and cpk13 mutants showed lower transcript levels of plant defensin gene PDF1.2 compared to wild-type plants. The CPK cascade was not directly linked to the herbivory-induced signaling pathways that were mediated by defense-related phytohormones such as jasmonic acid and ethylene. CPK3 was also suggested to be involved in a negative feedback regulation of the cytosolic Ca2+ levels after herbivory and wounding dama...
Heat shock response (HSR) generally plays a major role in sustaining protein homeostasis. In Escherichia coli, the activity and amount of the dedicated transcription factor σ(32) transiently increase upon heat shock. The initial induction... more
Heat shock response (HSR) generally plays a major role in sustaining protein homeostasis. In Escherichia coli, the activity and amount of the dedicated transcription factor σ(32) transiently increase upon heat shock. The initial induction is followed by chaperone-mediated negative feedback to inactivate and degrade σ(32). Previous work reported that signal recognition particle (SRP)-dependent targeting of σ(32) to the membrane is essential for feedback control, though how SRP recognizes σ(32) remained unknown. Extensive photo- and disulfide cross-linking studies in vivo now reveal that the highly conserved regulatory region of σ(32) that lacks a consecutive hydrophobic stretch interacts with the signal peptide-binding site of Ffh (the protein subunit of SRP). Importantly, the σ(32)-Ffh interaction observed was significantly affected by mutations in this region that compromise the feedback regulation, but not by deleting the DnaK/DnaJ chaperones. Homeostatic regulation of HSR thus re...