Biomedical subjects

R I Morimoto

Publications and source records attributed to R I Morimoto.

At least 55 records · Page 3Linked to original sources

PubMed1997

Repression of the heat shock factor 1 transcriptional activation domain is modulated by constitutive phosphorylation.

Heat shock transcription factor 1 (HSF1) is constitutively expressed in mammalian cells and negatively regulated for DNA binding and transcriptional activity. Upon exposure to heat shock and other forms of chemical and physiological stress, these activities of HSF1 are rapidly induced. In this report, we demonstrate that constitutive phosphorylation of HSF1 at serine residues distal to the transcriptional activation domain functions to repress transactivation. Tryptic phosphopeptide analysis of a collection of chimeric GAL4-HSF1 deletion and point mutants identified a region of constitutive phosphorylation encompassing serine residues 303 and 307. The significance of phosphorylation at serines 303 and 307 in the regulation of HSF1 transcriptional activity was demonstrated by transient transfection and assay of a chloramphenicol acetyltransferase reporter construct. Whereas the transfected wild-type GAL4-HSF1 chimera is repressed for transcriptional activity and derepressed by heat shock, mutation of serines 303 and 307 to alanine results in derepression to a high level of constitutive activity. Similar results were obtained with mutation of these serine residues in the context of full-length HSF1. These data reveal that constitutive phosphorylation of serines 303 and 307 has an important role in the negative regulation of HSF1 transcriptional activity at control temperatures.

3T3 Cells↗

PubMed1997

The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones.

Exposure of cells to stresses such as heat shock, oxidant injury and heavy metals causes an imbalance in protein metabolism which challenges the cell to respond rapidly, yet precisely, to minimize the deleterious effects of environmental and physiological stress. The heat-shock response, through the activation of HSFs, results in the elevated expression of heat-shock genes and the concomitant synthesis of HSPs and molecular chaperones. Molecular chaperones function in a variety of protein biosynthetic events and protect proteins from the deleterious effects of acute or chronic stress by stabilizing and refolding protein-folding intermediates or facilitating protein degradation. The accumulation of misfolded proteins has also become a central issue to diseases of protein folding, including sickle cell haemoglobin, cystic fibrosis and prion diseases, in addition to complex multifactorial diseases such as bacterial and viral infections, myocardial ischaemia, neurodegenerative diseases and cancer.

Animals↗

PubMed1996

Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23.

Molecular chaperones are essential proteins that participate in the regulation of steroid receptors in eukaryotes. The steroid aporeceptor complex contains the molecular chaperones Hsp90 and Hsp70, p48, the cyclophilin Cyp-40, and the associated proteins p23 and p60. In vitro folding assays showed that Cyp-40 and p23 functioned as molecular chaperones in a manner similar to that of Hsp90 or Hsp70. Although neither Cyp-40 nor p23 could completely refold an unfolded substrate, both proteins interacted with the substrate to maintain a nonnative folding-competent intermediate. Thus, the steroid aporeceptor complexes have multiple chaperone components that maintain substrates in an intermediate folded state.

Amino Acid Isomerases↗

PubMed1996

Modification of two distinct COOH-terminal domains is required for murine p53 activation by bacterial Hsp70.

Activation of the latent DNA binding function of human p53 protein by the bacterial Hsp70, DnaK, represents a unique reaction in which a heat shock protein can interact with a native protein to affect its function. We have localized a likely DnaK interaction site on native human p53 tetramers to a motif flanking the COOH-terminal casein kinase II and protein kinase C phosphorylation sites. Murine p53 is less efficiently activated by DnaK, which has permitted a search for factors that might cooperate in p53 activation by DnaK. We show that optimal activation by DnaK may be dependent upon the phosphorylation state of murine p53, in particular, modification of p53 at the cdc2 phosphorylation site by point mutation decreases the extent of activation by DnaK. Additionally, the monoclonal antibody PAb241, binding in the vicinity of the cdc2 phosphorylation site, is able to activate the specific DNA binding function of p53. This has led us to propose a second regulatory motif flanking the tetramerization domain of p53 that cooperates with factors binding at the negative regulatory domain in the extreme COOH terminus.

Amino Acid Sequence↗

PubMed1996

The human cytosolic molecular chaperones hsp90, hsp70 (hsc70) and hdj-1 have distinct roles in recognition of a non-native protein and protein refolding.

The properties of molecular chaperones in protein-assisted refolding were examined in vitro using recombinant human cytosolic chaperones hsp90, hsc70, hsp70 and hdj-1, and unfolded beta-galactosidase as the substrate. In the presence of hsp70 (hsc70), hdj-1 and either ATP or ADP, denatured beta-galactosidase refolds and forms enzymatically active tetramers. Interactions between hsp90 and non-native beta-galactosidase neither lead to refolding nor stimulate hsp70- and hdj-1-dependent refolding. However, hsp90 in the absence of nucleotide can maintain the non-native substrate in a 'folding-competent' state which, upon addition of hsp70, hdj-1 and nucleotide, leads to refolding. The refolding activity of hsp70 and hdj-1 is effective across a broad range of temperatures from 22 degrees C to 41 degrees C, yet at extremely low (4 degrees C) or high (>41 degrees C) temperatures refolding activity is reversibly inhibited. These results reveal two distinct features of chaperone activity in which a non-native substrate can be either maintained in a stable folding-competent state or refolded directly to the native state; first, that the refolding activity itself is temperature sensitive and second, that hsp90, hsp70 (hsc70) and hdj-1 each have distinct roles in these processes.

Adenine Nucleotides↗

PubMed1996

Activation of heat shock factor 1 DNA binding precedes stress-induced serine phosphorylation. Evidence for a multistep pathway of regulation.

Exposure of mammalian cells in culture to the anti-inflammatory drugs sodium salicylate or indomethacin results in activation of heat shock factor 1 (HSF1) DNA binding activity. We have previously shown that the drug-induced HSF1 becomes associated with the heat shock elements of the hsp70 promoter, yet transcription of the hsp70 gene is not induced (Jurivich, D. A., Sistonen, L., Kroes, R. A., and Morimoto, R. I. (1992) Science 255, 1243-1245). In this study, we have examined the basis for uncoupling the heat shock transcriptional response. Comparison of heat shock and drug-induced forms of HSF1 has revealed that the transcriptionally inert drug-induced HSF1 is constitutively but not inducibly serine-phosphorylated, whereas heat shock-induced HSF1 is both constitutively and inducibly serine-phosphorylated. The transcriptionally inert intermediate represented by drug-induced HSF1 can be converted to the transcriptionally active state by a subsequent exposure to heat shock. The only detectable change in HSF1 is the acquisition of inducible serine phosphorylation. These data reveal that acquisition of the trimeric DNA binding state of HSF1 is independent of and precedes inducible phosphorylation and furthermore that inducible phosphorylation correlates with transcriptional activation.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1996

Regulatory differences in the stress response of hippocampal neurons and glial cells after heat shock.

During periods of stress, cells depend on a transient, highly conserved, and regulated response to maintain homeostasis. This "heat shock response" is mediated transcriptionally by a multigene family of heat shock factors (HSF). The presence of multiple HSF suggests that activation of a given HSF is stress-specific. Using Western blot analysis, we have demonstrated the inability of primary cultured rat hippocampal neurons to induce a heat shock response after hyperthermia. In contrast, secondary cultured rat glial cells demonstrated a robust response. Examination of whole-cell extracts from the two cell types with gel shift mobility analysis and Western blot analysis revealed that although glial cells express HSF1 and HSF2, hippocampal neurons only express HSF2. Incubation of whole-cell extracts with monoclonal antisera raised against HSF1 and HSF2 before gel shift mobility analysis demonstrated HSF1 DNA-binding activity in glial cells and HSF2 DNA-binding activity in neurons. HSF1 has been shown to be the principal mediator of heat-induced heat shock gene expression. These results suggest that the deficient heat shock response of hippocampal neurons at this developmental stage is attributable to a lack of HSF1 expression. Furthermore, these results suggest that considerations of selective neuronal vulnerability to environmental stress should include the principal mediators of the stress response, the HSF.

Animals↗

PubMed1996

The transcriptional regulation of heat shock genes: a plethora of heat shock factors and regulatory conditions.

The inducible regulation of heat shock gene transcription is mediated by a family of heat shock factors (HSF) that respond to diverse forms of physiological and environmental stress including elevated temperature, amino acid analogs, heavy metals, oxidative stress, anti-inflammatory drugs, arachidonic acid, and a number of pathophysiological disease states. The vertebrate genome encodes a family of HSFs which are expressed ubiquitously, yet the DNA binding properties of each factor are negatively regulated and activated in response to specific conditions. This chapter will discuss the regulation of the HSF multi-gene family and the role of these transcriptional activators in the inducible expression of genes encoding heat shock proteins and molecular chaperones.

DNA-Binding Proteins↗

PubMed1996

Outlook.

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Cell Survival↗

PubMed1995

Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage.

The activation of heat shock genes by diverse forms of environmental and physiological stress has been implicated in a number of human diseases, including ischemic damage, reperfusion injury, infection, neurodegeneration, and inflammation. The enhanced levels of heat shock proteins and molecular chaperones have broad cytoprotective effects against acute lethal exposures to stress. Here, we show that the potent antiinflammatory drug indomethacin activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1). Perhaps relevant to its pharmacological use, indomethacin pretreatment lowers the temperature threshold of HSF1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves insufficient. The synergistic effect of indomethacin and elevated temperature is biologically relevant and results in the protection of cells against exposure to cytotoxic conditions.

DNA-Binding Proteins↗

PubMed1995

Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1.

The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. While the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rate is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a conformational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl-terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extreme carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70s. Deletion or mutation of EEVD affects the ATPase activity, the ability to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured firefly luciferase. Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp70 function and intermolecular interactions with HDJ-1.

Adenosine Triphosphatases↗

PubMed1995

Cloning and subcellular localization of human mitochondrial hsp70.

We report the cloning, nucleotide sequence, and localization of mitochondrial hsp70, a member of the human hsp70 multi-gene family. The human mthsp75 gene was cloned by screening an expression library with monoclonal antibody 3A3 that recognizes three members of the human hsp70 family (hsp70, hsc70, and a 75-kDa protein with characteristics identical to that previously established for mitochondrial hsp70). The identity of the 75-kDa protein was confirmed by subcellular fraction of HeLa cells and the demonstration that the 3A3-reactive 75-kDa protein co-fractionates with mitochondrial localized proteins. The nucleotide sequence of the respective cDNA clone revealed an open reading frame of 679 amino acids with extensive sequence identity with members of the human hsp70 family. The derived amino-terminal pre-sequence shares features common to other mitochondrial targeting sequences. The identity of the cDNA was unequivocally established by introduction of an epitope-tag at the carboxyl terminus of the cloned gene, transfection and analysis by immunofluorescence. The tagged 75-kDa protein localizes to mitochondria, thus providing conclusive evidence that it corresponds to the human mitochondrial hsp70, referred to here as mthsp75.

Amino Acid Sequence↗

PubMed1995

The DNA-binding properties of two heat shock factors, HSF1 and HSF3, are induced in the avian erythroblast cell line HD6.

Avian cells express three heat shock transcription factor (HSF) genes corresponding to a novel factor, HSF3, and homologs of mouse and human HSF1 and HSF2. Analysis of the biochemical and cell biological properties of these HSFs reveals that HSF3 has properties in common with both HSF1 and HSF2 and yet has features which are distinct from both. HSF3 is constitutively expressed in the erythroblast cell line HD6, the lymphoblast cell line MSB, and embryo fibroblasts, and yet its DNA-binding activity is induced only upon exposure of HD6 cells to heat shock. Acquisition of HSF3 DNA-binding activity in HD6 cells is accompanied by oligomerization from a non-DNA-binding dimer to a DNA-binding trimer, whereas the effect of heat shock on HSF1 is oligomerization of an inert monomer to a DNA-binding trimer. Induction of HSF3 DNA-binding activity is delayed compared with that of HSF1. As occurs for HSF1, heat shock leads to the translocation of HSF3 to the nucleus. HSF exhibits the properties of a transcriptional activator, as judged from the stimulatory activity of transiently overexpressed HSF3 measured by using a heat shock element-containing reporter construct and as independently assayed by the activity of a chimeric GAL4-HSF3 protein on a GAL4 reporter construct. These results reveal that HSF3 is negatively regulated in avian cells and acquires DNA-binding activity in certain cells upon heat shock.

Animals↗

PubMed1995

The carboxyl-terminal transactivation domain of heat shock factor 1 is negatively regulated and stress responsive.

We have characterized a stress-responsive transcriptional activation domain of mouse heat shock factor 1 (HSF1) by using chimeric GAL4-HSF1 fusion proteins. Fusion of the GAL4 DNA-binding domain to residues 124 to 503 of HSF1 results in a chimeric factor that binds DNA yet lacks any transcriptional activity. Transactivation is acquired upon exposure to heat shock or by deletion of a negative regulatory domain including part of the DNA-binding-domain-proximal leucine zippers. Analysis of a collection of GAL4-HSF1 deletion mutants revealed the minimal region for the constitutive transcriptional activator to map within the extreme carboxyl-terminal 108 amino acids, corresponding to a region rich in acidic and hydrophobic residues. Loss of residues 395 to 425 or 451 to 503, which are located at either end of this activation domain, severely diminished activity, indicating that the entire domain is required for transactivation. The minimal activation domain of HSF1 also confers enhanced transcriptional response to heat shock or cadmium treatment. These results demonstrate that the transcriptional activation domain of HSF1 is negatively regulated and that the signal for stress induction is mediated by interactions between the amino-terminal negative regulator and the carboxyl-terminal transcriptional activation domain.

3T3 Cells↗

PubMed1994

Effects of neurohormonal stress and aging on the activation of mammalian heat shock factor 1.

The mammalian heat shock response has been investigated extensively using tissue culture cells with only a limited amount of information available on animals and intact tissues. The neurohormonal stress response mediated by the hypothalamic-pituitary-adrenal axis leads to the activation of heat shock factor (HSF) in rat adrenal tissue. Here we show through the use of antibodies specific to each member of the HSF family that restraint-induced stress in intact Wistar rats and adrenocorticotropic hormone treatment of hypophysectomized animals leads to the activation of HSF1 monomers to trimers with DNA-binding activity. Because HSF1 is also the target factor for metabolic and environmental stress, these data reveal an intersection of pathways leading to HSF1 activation. Comparison of the biochemical properties and levels of HSF1 in the Wistar and Fischer 344 rat strains reveals that HSF1 is constitutively present in an activated DNA-binding state in the adrenals of Fischer 344 rats. During aging, the levels of HSF1 remain constant, yet the transcription factor from aged animals exhibits a decreased ability to bind DNA.

Adrenal Glands↗

PubMed1994

Small subclass of rat olfactory neurons with specific bulbar projections is reactive with monoclonal antibodies to the HSP70 heat shock protein.

As part of a study of turnover of rat olfactory receptor neurons we have been examining immunohistochemical expression of members of the 70 kD heat shock protein (HSP70) family in the olfactory epithelium. Expression of HSP70 family members is up-regulated in many cells following exposure to physiologically stressing conditions. Because dying neurons are likely to undergo some sort of physiological stress before the onset of frank degeneration, we hoped that anti-HSP70 monoclonal antibodies would prove to be useful markers for early stages of olfactory neuron cell death. Two anti-human HSP70 monoclonal antibodies were used, Mabs 2A4 and 3a3. Two-dimensional gel electrophoresis/western blot analysis indicates that these Mabs are reactive with the HSC70 and HSP70 members of the rat HSP70 family. Immunohistological observations show that both Mabs are strongly reactive with a widely dispersed subpopulation of olfactory receptor neurons. Morphological, immunohistological, and autoradiographic birthdating analyses demonstrate that reactive cells are fully mature receptor neurons. Their reactivity, however, does not appear to be stress-related. More significantly, axons of reactive neurons show intense anti-2A4 reactivity. This has allowed us to trace these axons to their target glomeruli in the olfactory bulb, demonstrating that the reactive neurons project to just one to two glomeruli on either side of each bulb via consistent and predictable pathways. This is the first subpopulation of olfactory receptor neurons to be traced to such a small number of glomeruli. Given this extremely small number, it seems likely that the reactive receptor cell subpopulation serves some specific olfactory function. In addition, axonal 2A4 reactivity should also prove useful in defining the relative roles of receptor neurons and glomeruli in the establishment of epithelial-glomerular connections.

Animals↗

PubMed1994

Deficient induction of human hsp70 heat shock gene transcription in Y79 retinoblastoma cells despite activation of heat shock factor 1.

One of the basic features of the inducible heat shock response is the activation of heat shock factor which results in the rapid transcriptional induction of the heat shock genes. Although it is widely considered that the heat shock response is ubiquitous, several reports have indicated that the transcriptional response can vary in both intensity and kinetics and often in a tissue-specific manner. Of interest have been studies on the expression of heat shock genes in the brain, particularly observations that certain cultured neuronal cells exhibit a diminished heat shock response. We demonstrate that transcription of the gene encoding a 70-kDa heat shock protein (hsp70) is diminished upon heat shock in Y79 human retinoblastoma cells (which are of neuronal origin) despite both the activation of heat shock factor 1 and induced transcription of another heat shock gene, hsp90 alpha. This uncoupling of stress-induced transcription of the hsp70 and hsp90 alpha genes, which are typically coordinately regulated in response to stress, appears to be due to the selective inability of trans-acting factors, including heat shock factor 1, to bind in vivo to the hsp70 promoter as the result of a chromatin-mediated effect.

DNA-Binding Proteins↗

PubMed1994

c-Myb-induced trans-activation mediated by heat shock elements without sequence-specific DNA binding of c-Myb.

The c-myb proto-oncogene product (c-Myb) can transactivate the human hsp70 promoter in a transient cotransfection assay. The present studies have demonstrated that the heat shock element (HSE) in the hsp70 promoter mediates trans-activation by c-Myb. Mutagenesis of the DNA sequence in HSE indicated that the NGAAN motif is necessary for not only the heat shock response but also the c-Myb-induced trans-activation. The HSE in the hsp70 promoter does not contain a c-Myb-binding site, implying that the sequence-specific DNA binding of c-Myb is not required for the HSE-dependent trans-activation by c-Myb. We had demonstrated that a disruption of the leucine zipper motif in the central portion of the c-Myb molecule increased the degree of c-Myb-induced trans-activation of the promoter containing c-Myb-binding sites, suggesting that a putative inhibitor binds to c-Myb through this leucine zipper (Kanie-Ishii, C., MacMillan, E. M., Nomura, T., Sarai, A., Ramsay, R. G., Aimoto, S., Ishii, S., and Gonda, T. J. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3088-3092). However, disruption of the leucine zipper in c-Myb abolished the HSE-dependent trans-activation by c-Myb, whereas deletion of the transcriptional activation domain containing acidic amino acids in c-Myb did not abolish the HSE-dependent trans-activation by c-Myb. These results suggest that c-HSEs by interacting with unidentified trans-acting factor(s) but not by a direct binding to the promoter through its DNA-binding domain.

Animals↗