Nick Ovsenek

HSP90 Interacts with and Regulates the Activity of Heat Shock Factor 1 in Xenopus Oocytes

ABSTRACT Transcriptional activation of heat shock genes is a reversible and multistep process involving conversion of inactive heat shock factor 1 (HSF1) monomers into heat shock element (HSE)-binding homotrimers, hyperphosphorylation, and further modifications that induce full transcriptional competence. HSF1 is controlled by multiple regulatory mechanisms, including suppression by additional cellular factors, physical interactions with HSP70, and integration into different cellular signaling cascades. However, the signaling mechanisms by which cells respond to stress and control the HSF1 activation-deactivation pathway are not known. Here we demonstrate that HSP90, a cellular chaperone known to regulate several signal transduction molecules and transcription factors, functions in the regulation of HSF1. The existence of HSF1-HSP90 heterocomplexes was shown by coimmunoprecipitation of HSP90 with HSF1 from unshocked and heat-shocked nuclear extracts, recognition of HSF1-HSE complexes in vitro by using HSP90 antibodies (Abs), and recognition of HSF1 in vivo by HSP90 Abs microinjected directly into oocyte nuclei. The functional impact of HSP90-HSF1 interactions was analyzed by using two strategies: direct nuclear injection of HSP90 Abs and treatment of cells with geldanamycin (GA), an agent that specifically blocks the chaperoning activity of HSP90. Both HSP90 Abs and GA delayed the disassembly of HSF1 trimers during recovery from heat shock and specifically inhibited heat-induced transcription from a chloramphenicol acetyltransferase reporter construct under control of the hsp70 promoter. HSP90 Abs activated HSE binding in the absence of heat shock, an effect that could be reversed by subsequent injection of purified HSP90. GA did not activate HSE binding under nonshock conditions but increased the quantity of HSE binding induced by heat shock. On the basis of these findings and the known properties of HSP90, we propose a new regulatory model in which HSP90 participates in modulating HSF1 at different points along the activation-deactivation pathway, influencing the interconversion between monomeric and trimeric conformations as well as transcriptional activation. We also put forth the hypothesis that HSP90 links HSF1 to cellular signaling molecules coordinating the stress response.

Multiple Components of the HSP90 Chaperone Complex Function in Regulation of Heat Shock Factor 1 In Vivo

ABSTRACT Rapid and transient activation of heat shock genes in response to stress is mediated in eukaryotes by the heat shock transcription factor HSF1. It is well established that cells maintain a dynamic equilibrium between inactive HSF1 monomers and transcriptionally active trimers, but little is known about the mechanism linking HSF1 to reception of various stress stimuli or the factors controlling oligomerization. Recent reports have revealed that HSP90 regulates key steps in the HSF1 activation-deactivation process. Here, we tested the hypothesis that components of the HSP90 chaperone machine, known to function in the folding and maturation of steroid receptors, might also participate in HSF1 regulation. Mobility supershift assays using antibodies against chaperone components demonstrate that active HSF1 trimers exist in a heterocomplex with HSP90, p23, and FKBP52. Functional in vivo experiments in Xenopus oocytes indicate that components of the HSF1 heterocomplex, as well as other components of the HSP90 cochaperone machine, are involved in regulating oligomeric transitions. Elevation of the cellular levels of cochaperones affected the time of HSF1 deactivation during recovery: attenuation was delayed by immunophilins, and accelerated by HSP90, Hsp/c70, Hip, or Hop. In immunotargeting experiments with microinjected antibodies, disruption of HSP90, Hip, Hop, p23, FKBP51, and FKBP52 delayed attenuation. In addition, HSF1 was activated under nonstress conditions after immunotargeting of HSP90 and p23, evidence that these proteins remain associated with HSF1 monomers and function in their repression in vivo. The remarkable similarity of HSF1 complex chaperones identified here (HSP90, p23, and FKBP52) and components in mature steroid receptor complexes suggests that HSF1 oligomerization is regulated by a foldosome-type mechanism similar to steroid receptor pathways. The current evidence leads us to propose a model in which HSF1, HSP90 and p23 comprise a core heterocomplex required for rapid conformational switching through interaction with a dynamic series of HSP90 subcomplexes.

Preferential activation of HSF-binding activity and hsp70 gene expression in Xenopus heart after mild hyperthermia

We have examined the effect of mild hyperthermia on the pattern of heat shock transcription factor (HSF) binding activity, heat shock protein 70 (hsp70) and hsp30 gene expression and protein denaturation in selected tissues of adult Xenopus namely, heart, hind limb muscle, eye, liver and spleen. In these studies it was found that heart tissue was the most thermally sensitive of all of the tissues examined since maintenance of adult frogs at 26 degrees C resulted in a preferential activation of HSF binding. Thus, heart has a lowered set point temperature for HSF activation compared to the other tissues examined. At 30 degrees C HSF activation was observed in all of the tissues examined. Heart HSF activation at 26 degrees C was correlated with an increase in hsp70 mRNA and Hsp70 protein accumulation. At 28 degrees C the largest amount of hsp70 and hsp30 mRNA accumulation was detected in heart and skeletal muscle compared to other tissues while hsp70 mRNA accumulation was relatively low in spleen and hsp30 mRNA accumulation was not detectable in eyes, liver and spleen. Incubation of adult frogs at 30 degrees C resulted in enhanced hsp70 and hsp30 mRNA accumulation in all of the tissues. Finally, we have used differential scanning calorimetry (DSC) to compare the temperatures at which protein denaturation occurs in heart and liver tissue. The onset of protein denaturation (T0) occurred approximately 8.5 degrees C lower in heart compared to liver. Also the midpoint of the DSC profile (T1/2) was approximately 10.4 degrees C lower in heart than in liver. Thus, heart proteins are generally more thermolabile than proteins in liver tissue. Taken together these data suggest that heart is more sensitive than the other tissues examined with respect to moderate increases in environmental temperature.

Xenopus heat shock factor 1 is a nuclear protein before heat stress.

Stress-induced expression of the heat shock (hs) genes in eukaryotes is mediated by a transcription factor known as heat shock factor 1 (HSF1). HSF1 is present in a latent, monomeric form in unstressed metazoan cells and upon exposure to heat or other forms of stress is converted to an “active” trimeric form, which binds the promoters of hs genes and induces their transcription. The conversion of HSF1 to its active form is hypothesized to be a multistep process involving (i) oligomerization of HSF1, plus (ii) additional changes in its physical conformation, (iii) changes in its phosphorylation state, and for some species (iv) translocation from the cytoplasm to the nucleus. Oligomerization of HSF appears to be essential for high affinity DNA binding, but it remains unclear whether the other steps occur in all organisms or what their mechanistic roles are. In this study we have examined if heat-induced cytoplasmic-nuclear translocation of HSF1 occurs in Xenopus oocytes. We observed that germinal vesicles (nuclei) that were physically dissected from unshocked Xenopus laevis oocytes contain no HSF1 binding activity. Interestingly, in vitro heat shock treatments of isolated nuclei from unshocked oocytes activated HSF1 binding, indicating that HSF1 must have been present in the unshocked nuclei prior to isolation. Induction of HSF1 binding was not observed in enucleated oocytes. Western blot analysis using an affinity-purified polyclonal antibody made against X. laevis HSF1 showed that HSF1 is present in equal amounts in unshocked and shocked oocytes and isolated nuclei. HSF1 was not detected in enucleated oocytes. These results clearly demonstrate that HSF1 is a nuclear protein in oocytes prior to exposure to stress. In Xenopus oocytes, therefore, HSF1 translocation from the cytoplasm to the nucleus is not part of the multistep process of HSF1 activation. These results also imply that the signals and/or factors involved in HSF1 activation must have their effect in the nuclear compartment.

INDUCTION OF THE DNA-BINDING AND TRANSCRIPTIONAL ACTIVITIES OF HEAT SHOCK FACTOR 1 IS UNCOUPLED IN XENOPUS OOCYTES

The DNA-binding and transcriptional activities of the heat shock transcription factor 1 (HSF1) are repressed under normal conditions and rapidly upregulated by heat stress. Here, we tested for the ability of various stress agents to activate HSF1 in the Xenopus oocyte model system. The HSE-binding activity of HSF1 was induced by a number of chemical stresses including cadmium, aluminum, iron, mercury, arsenite, ethanol, methanol, and salicylate. HSE-binding was not induced by several stresses known to induce the synthesis of hsps in other cell types in different organisms including zinc, copper, cobalt, manganese, recovery from anoxia, UV-irradiation, and increased pH. The inability of several known inducers of the stress response to activate the HSE-binding ability of HSF1 suggests that certain stress activation pathways may be absent or inactive in oocytes. The transcriptional activity of oocyte HSF1 was induced by heat, cadmium, and arsenite, but many of the agents that induced HSE-binding failed to stimulate HSF1-mediated transcription. The apparent uncoupling of inducible HSE-binding and transcriptional activities of HSF1 under a variety of stress regimes indicates that these events are regulated by independent mechanisms in the oocyte.

Assembly of the Yin Yang 1 transcription factor into messenger ribonucleoprotein particles requires direct RNA binding activity.

The early stages of vertebrate development depend heavily on control of maternally transcribed mRNAs that are stored for long periods in complexes termed messenger ribonucleoprotein particles (mRNPs) and utilized selectively following maturation and fertilization. The transcription factor Yin Yang 1 (YY1) is associated with cytoplasmic mRNPs in vertebrate oocytes; however, the mechanism by which any of the mRNP proteins associate with mRNA in the oocyte is unknown. Here we demonstrate the mechanism by which YY1 associates with mRNPs depends on its direct RNA binding activity. High affinity binding for U-rich single-stranded RNA and A:U RNA duplexes was observed in the nanomolar range, similar to the affinity for the cognate double-stranded DNA-binding element. Similar RNA binding affinity was observed with endogenous YY1 isolated from native mRNP complexes. In vivo expression experiments reveal epitope-tagged YY1 assembled into high molecular mass mRNPs, and assembly was blocked by microinjection of high affinity RNA substrate competitor. These findings present the first clues to how mRNPs assemble during early development.

Asymmetric distribution of metals in the Xenopus laevis oocyte: a synchrotron X-ray fluorescence microprobe study

The asymmetric distribution of many components of the Xenopus oocyte, including RNA, proteins, and pigment, provides a framework for cellular specialization during development. During maturation, Xenopus oocytes also acquire metals needed for development, but apart from zinc, little is known about their distribution. Synchrotron X-ray fluorescence microprobe was used to map iron, copper, and zinc and the metalloid selenium in a whole oocyte. Iron, zinc, and copper were asymmetrically distributed in the cytoplasm, while selenium and copper were more abundant in the nucleus. A zone of high copper and zinc was seen in the animal pole cytoplasm. Iron was also concentrated in the animal pole but did not colocalize with zinc, copper, or pigment accumulations. This asymmetry of metal deposition may be important for normal development. Synchrotron X-ray fluorescence microprobe will be a useful tool to examine how metals accumulate and redistribute during fertilization and embryonic development.

Troglitazone reduces heat shock protein 70 content in primary rat hepatocytes by a ubiquitin proteasome independent mechanism

Troglitazone (TRG) is an antidiabetic agent that increases the insulin sensitivity of target tissues in non-insulin-dependent diabetes mellitus. Therapy with troglitazone has been associated with severe hepatic injury in a small percentage of patients and the mechanism of TRG-induced hepatotoxicity remains unclear. A family of highly conserved stress proteins identified as heat shock proteins (Hsps), are well-known to protect cells against a wide variety of toxic conditions such as extreme temperature changes, oxidative stress and toxic drugs. The stress-inducible Hsp 70 protein is one of the best-known endogenous factors protecting cells from injury under various stress conditions. Here we examined the effects of TRG on Hsp 70 mRNA and protein expression in primary cultures of rat hepatocytes. We also investigated the effects of TRG in an in vivo model by examining Hsp 70 protein levels in livers prepared from C57 mice fed a 0.2% dietary admixture of TRG. Levels of Hsp 70 mRNA increased in a concentration-dependent manner in rat hepatocytes treated for 8h with increasing concentrations of TRG. However, Hsp 70 protein levels decreased significantly in cells treated with increasing concentrations of TRG. C57 mice fed a 0.2% admixture of TRG for 10 days, also demonstrated decreased liver Hsp 70 protein levels. To investigate whether TRG decreased Hsp 70 protein levels by activating the ubiquitin-proteasome pathway, cells were pretreated with 10 microM lactacystin, a potent and specific inhibitor of this pathway. Lactacystin pretreatment failed to prevent TRG-induced decrease in Hsp 70 protein. The data suggests that TRG-induced effects may be mediated through another system of regulated proteolysis or may involve a post-transcriptional regulator mechanism. The mechanism of TRG-induced hepatotoxicity remains unclear, however, the effects induced by TRG on Hsp 70 may, in part, play a role.

Constitutive and heat-inducible heat shock element binding activities of heat shock factor in a group of filamentous fungi.

This study represents the initial characterization of the heat shock factor (HSF) in filamentous fungi. We demonstrate that HSFs from Beauveria bassiana, Metarhizium anisopliae, Tolypocladium nivea, Paecilomyces farinosus, and Verticillium lecanii bind to the heat shock element (HSE) constitutively (non-shocked), and that heat shock resulted in increased quantities and decreased mobility of HSF-HSE complexes. The monomeric molecular mass of both heat-induced and constitutive HSFs was determined to be 85.8 kDa by UV-crosslinking and the apparent molecular masses of the native HSF-HSE complexes as determined by pore exclusion gradient gel electrophoresis was 260 and 300 kDa, respectively. Proteolytic band clipping assays using trypsin and chymotrypsin revealed an identical partial cleavage profile for constitutive and heat-induced HSF-HSE complexes. Thus, it appears that both constitutive and heat-inducible complexes are formed by trimers composed of the same HSF molecule which undergoes conformational changes during heat shock. The mobility difference between the complexes was not abolished by enzymatic dephosphorylation and deglycosylation, indicating that the reduced mobility of the heat-induced HSF is probably due to a post-translational modification other than phosphorylation or glycosylation.

Parameters for effective in vitro production of zinc finger nucleic acid‐binding proteins

Immobilized metal affinity chromatography (IMAC) is widely used for the production of recombinant proteins for a variety of applications; however, a number of challenges are typically encountered by researchers depending on the properties of the specific proteins in question. Here, we describe technical issues we have encountered in production of recombinant zinc finger nucleic acid‐binding proteins by IMAC intended for detailed and accurate in vitro analysis. The process encountered leading to a modified IMAC protocol for effective production of high‐purity, native zinc finger nucleic acid‐binding proteins is described in detail. The parameters with respect to solubility, lysis and redox conditions, removal of residual metal ions with chelating agents, and renaturation in the presence of divalent metal cations are described. These procedures have been extended to production of a wide array of RNA‐binding proteins in our laboratory and would be relevant to a number of protein purification applications.