Calmodulin -regulated processes and intracellular calcium in the heat stress response of mammalian cells

Three approaches were used to examine the role of Ca$\sp{2+}$- and/or calmodulin (CaM)-regulated processes in the mammalian heat stress response. The focus of the first approach was on the major Ca$\sp{2+}$-binding protein, CaM, and involved the use of CaM antagonists that perturbed CaM-regulated processes during heat stress. The second approach involved the use of a cell line and its BPV-1 transformants that express increased basal levels of CaM, or parvalbumin--a Ca$\sp{2+}$-binding protein not normally found in these cells. The last approach used Ca$\sp{2+}$ chelators to buffer Ca$\sp{2+}$-transients.^ The principle conclusions resulting from these three experimental approaches are: (1) CaM antagonists cause a temperature-dependent potentiation of heat killing, but do not inhibit the triggering and development of thermotolerance suggesting some targets for heat killing are different from those that lead to thermotolerance; (2) Members of major HSP families (especially HSP70) can bind to CaM in a Ca$\sp{2+}$-dependent manner in vitro, and HSP have been associated with events leading to thermotolerance. But, because thermotolerance is not affected by CaM antagonists, and antagonists should interfere with HSP binding to CaM, the events leading to triggering or developing thermotolerance were not strongly dependent on HSP binding to CaM; (3) CaM antagonists can also bind to HSP70 (and possibly other HSP) suggesting an alternative mechanism for the action of these agents in heat killing may involve direct binding to other proteins, like HSP70, whose function is important for survival following heating and inhibiting their activity; and (4) The signal governing the rate of synthesis of another major HSP group, the HSP26 family, can be largely abrogated by elevated Ca$\sp{2+}$-binding proteins or Ca$\sp{2+}$ chelators without significantly reducing survival or thermotolerance suggesting if the HSP26 family is involved in either end point, it may function in (Ca$\sp{2+}$) $\sb{\rm i}$ homeostasis. ^

Investigations of stress responses in Saccharomyces cerevisiae: Transcriptional control and heat shock protein function

The baker's yeast, Saccharomyces cerevisiae responds to the cytotoxic effects of elevated temperature (37-42°C) by activating transcription of ∼150 genes, termed heat shock genes, collectively required to compensate for the abundance of misfolded and aggregated proteins and various physiological modifications necessary for the cell to survive and grow at heat shock temperatures. An intriguing facet of the yeast heat shock response is the remarkable similarity it shares with the global remodeling that occurs in mammalian cells in response to numerous pathophysiological conditions including cancer and cardiovascular disease and thus provides an ideal model system. I have therefore investigated several novel features of stress signaling, transcriptional regulation, and physiology. Initial work focused on the characterization of SYM1, a novel heat shock gene in yeast which was demonstrated to be required for growth on the nonfermentable carbon source ethanol at elevated temperature, and to be the functional ortholog of the mammalian kidney disease gene, Mpv17. Additional work addressed the role of two proteins, the Akt-related kinase, Sch9, and Sse1, the yeast Hsp110 protein chaperone homolog, in signaling by protein kinase A, establishing Sse1 as a critical negative regulator of this pathway. Furthermore, I have demonstrated a role for Sse1 in biogenesis and stability of the stress-response transcription factor, Msn2; a finding that has been extended to include a select subset of additional high molecular weight proteins, suggesting a more global role for this chaperone in stabilizing the cellular proteome. The final emphasis of my doctoral work has included the finding that celastrol, a compound isolated from the plant family Celasfraceae, a component of traditional Chinese herbal medicine, can activate heat shock transcription factor (Hsf1) in yeast and mammalian cells through an oxidative stress mechanism. Celastrol treatment simultaneously activates both heat shock and oxidative stress response pathways, resulting in increased cytoprotection. ^

Stress-induced targeting of molecular chaperones in the yeast Saccharomyces cerevisiae

The eukaryotic stress response is an essential mechanism that helps protect cells from a variety of environmental stresses. Cell death can result if cells are not able to properly adapt and protect themselves against adverse stress conditions. Failure to properly deal with stress has implications in human diseases including neurodegenerative disorders and distinct cancers, emphasizing the importance of understanding the eukaryotic stress response in detail. As part of this response, expression of a battery of heat shock proteins (HSP) is induced, which act as molecular chaperones to assist in the repair or triage of unfolded proteins. The 90-kDa HSP (Hsp90) operates in the context of a multi-chaperone complex to promote the maturation of nuclear and cytoplasmic clients. I have discovered that Hsp90 and the co-chaperone Sba1 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells in a karyopherin-dependent manner. I isolated nuclear accumulation- defective HSP82 mutant alleles to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to prevent nuclear accumulation of Hsp90 in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele, further linking localization to Hsp90 functional status. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program. The yeast molecular chaperone Hsp104 is a member of the Hsp100 superfamily of AAA+ ATPases. Unlike the Hsp90 family of chaperones, Hsp104 is not restricted to a specific set of client proteins, but rather assists in reactivating stress-denatured proteins by solubilizing protein aggregates. I have discovered that Hsp104, along with the Hsp70 chaperone, Ssa1, and the sHSP Hsp26 accumulate into RNA processing bodies (P- bodies) and stress granules, sites of mRNA metabolism. I found that Hsp104 recruits both Ssa1 and Hsp26 to P-bodies and that these three chaperones are required for stress granule formation. These findings suggest a possible role for chaperones in mRNA metabolism by aiding in the assembly, disassembly or conversion of these enigmatic mRNP complexes. Taken together, the work presented in this dissertation serves to better understand the eukaryotic stress response by illustrating the importance of subcellular-chaperone localization in key biological processes.

Activation of heat shock and antioxidant responses by the natural product celastrol: transcriptional signatures of a thiol-targeted molecule.

Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product.

Evaluation of oxidative damage and renal distal tubule cell stress response following exposure to lindane

Lindane, or γ-hexachlorocyclohexane, is a chlorinated hydrocarbon pesticide that was banned from U.S. production in 1976, but until recently continued to be imported and applied for occupational and domestic purposes. Lindane is known to cause central nervous system (CNS), immune, cardiovascular, reproductive, liver, and kidney toxicity. The mechanism for which lindane interacts with the CNS has been elucidated, and involves antagonism of the γ-aminobutyric acid/benzodiazepine (GABAA/BZD) receptor. Antagonism of this receptor results in the inhibition of Cl- channel flux, with subsequent convulsions, seizures, and paralysis. This response makes lindane a desirable defense against arthropod pests in agriculture and the home. However, formulation and application of this compound can contribute to human toxicity. In conjunction with this exposure scenario, workers may be subject to both heat and physical stress that may increase their susceptibility to pesticide toxicity by altering their cellular stress response. The kidneys are responsible for maintaining osmotic homeostasis, and are exposed to agents that undergo urinary excretion. The mechanistic action of lindane on the kidneys is not well understood. Lindane, in other organ systems, has been shown to cause cellular damage by generation of free radicals and oxidative stress. Previous research in our laboratory has shown that lindane causes apoptosis in distal tubule cells, and delays renal stress response under hypertonic stress. Characterizing the mechanism of action of lindane under conditions of physiologic stress is necessary to understand the potential hazard cyclodiene pesticides and other organochlorine compounds pose to exposed individuals under baseline conditions, as well as under conditions of physiologic stress. We demonstrated that exposure to lindane results in oxidative damage and dysregulation of glutathione response in renal distal tubule (MDCK) cells. We showed that under conditions of hypertonic stress, lindane-induced oxidative stress resulted in early onset apoptosis and corresponding down-regulated expression of the anti-apoptotic protein, Bcl-xL. Thus, the interaction of lindane with renal peripheral benzodiazepine receptors (PBR) is associated with attenuation of cellular protective proteins, making the cell more susceptible to injury or death. ^

Differential stress response and susceptibility to oxidative injury in alveolar macrophages from C57BL/6J and C3H/HeJ mice following oxidant exposure

Studies have demonstrated a variable response to ozone among individuals and animal species and strains. For instance, C57BL/6J mice have a greater inflammatory response to ozone exposure than C3H/HeJ mice. In these studies, I utilized these strain differences in an effort to derive a mechanistic explanation to the variable strain sensitivity to ozone exposure. Therefore, alveolar macrophages (AM) from C57BL/6J and C3H/HeJ mice were exposed in vitro to hydrogen peroxide ($\rm H\sb2O\sb2$), heat and acetyl ceramide or in vivo to ozone. Necrosis and DNA fragmentation in macrophages from the two murine strains were determined to assess cytotoxicity following these treatments. In addition, synthesis and expression of the stress proteins, stress protein 72 (SP72) and heme oxygenase (HO-1), were examined following treatments. The in vitro experiments were conducted to eliminate the possibility of in vivo confounders (i.e., differences in breathing rates in the two strains) and thus directly implicate some inherent difference between cells from the two murine strains. $\rm H\sb2O\sb2$ and heat caused greater cytotoxicity in AM from C57BL/6J than C3H/HeJ mice and DNA fragmentation was a particularly sensitive indicator of cell injury. Similarly, AM from C57BL/6J mice were more sensitive to ozone exposure than cells from C3H/HeJ mice. Exposure to either 1 or 0.4 ppm ozone caused greater cytotoxicity in macrophages from C57BL/6J mice compared to macrophages from C3H/HeJ mice. The increased sensitivity of AM to injury was associated with decreased synthesis and expression of stress proteins. AM from C57BL/6J mice synthesized and expressed significantly less stress proteins in response to heat and ozone than AM from C3H/HeJ mice. Heat treatment resulted in greater synthesis and expression of SP72. In addition, macrophages from C57BL/6J mice expressed lower amounts of HO-1 than macrophages from C3H/HeJ mice following 0.4 ppm ozone exposure. Therefore, AM from C57BL/6J mice are more susceptible to oxidative injury than AM from C3H/HeJ mice which might be due to differential expression of stress proteins in these cells. ^

Regulation of the heat shock response by thiol-reactive compounds in the yeast Saccharomyces cerevisiae

Cells govern their activities and modulate their interactions with the environment to achieve homeostasis. The heat shock response (HSR) is one of the most well studied fundamental cellular responses to environmental and physiological challenges, resulting in rapid synthesis of heat shock proteins (HSPs), which serve to protect cellular constituents from the deleterious effects of stress. In addition to its role in cytoprotection, the HSR also influences lifespan and is associated with a variety of human diseases including cancer, aging and neurodegenerative disorders. In most eukaryotes, the HSR is primarily mediated by the highly conserved transcription factor HSF1, which recognizes target hsp genes by binding to heat shock elements (HSEs) in their promoters. In recent years, significant efforts have been made to identify small molecules as potential pharmacological activators of HSF1 that could be used for therapeutic benefit in the treatment of human diseases relevant to protein conformation. However, the detailed mechanisms through which these molecules drive HSR activation remain unclear. In this work, I utilized the baker's yeast Saccharomyces cerevisiae as a model system to identify a group of thiol-reactive molecules including oxidants, transition metals and metalloids, and electrophiles, as potent activators of yeast Hsf1. Using an artificial HSE-lacZ reporter and the glucocorticoid receptor system (GR), these diverse thiol-reactive compounds are shown to activate Hsf1 and inhibit Hsp90 chaperone complex activity in a reciprocal, dose-dependent manner. To further understand whether cells sense these reactive compounds through accumulation of unfolded proteins, the proline analog azetidine-2-carboxylic acid (AZC) and protein cross-linker dithiobis(succinimidyl propionate) (DSP) were used to force misfolding of nascent polypeptides and existing cytosolic proteins, respectively. Both unfolding reagents display kinetic HSP induction profiles dissimilar to those generated by thiol-reactive compounds. Moreover, AZC treatment leads to significant cytotoxicity, which is not observed in the presence of the thiol-reactive compounds at the concentrations sufficient to induce Hsf1. Additionally, DSP treatment has little to no effect on Hsp90 functions. Together with the ultracentrifugation analysis of cell lysates that detected no insoluble protein aggregates, my data suggest that at concentrations sufficient to induce Hsf1, thiol-reactive compounds do not induce the HSR via a mechanism based on accumulation of unfolded cytosolic proteins. Another possibility is that thiol-reactive compounds may influence aspects of the protein quality control system such as the ubiquitin-proteasome system (UPS). To address this hypothesis, β-galactosidase reporter fusions were used as model substrates to demonstrate that thiol-reactive compounds do not inhibit ubiquitin activating enzymes (E1) or proteasome activity. Therefore, thiol-reactive compounds do not activate the HSR by inhibiting UPS-dependent protein degradation. I therefore hypothesized that these molecules may directly inactivate protein chaperones, known as repressors of Hsf1. To address this possibility, a thiol-reactive biotin probe was used to demonstrate in vitro that the yeast cytosolic Hsp70 Ssa1, which partners with Hsp90 to repress Hsf1, is specifically modified. Strikingly, mutation of conserved cysteine residues in Ssa1 renders cells insensitive to Hsf1 activation by cadmium and celastrol but not by heat shock. Conversely, substitution with the sulfinic acid and steric bulk mimic aspartic acid led to constitutive activation of Hsf1. Cysteine 303, located in the nucleotide-binding/ATPase domain of Ssa1, was shown to be modified in vivo by a model organic electrophile using Click chemistry technology, verifying that Ssa1 is a direct target for thiol-reactive compounds through adduct formation. Consistently, cadmium pretreatment promoted cells thermotolerance, which is abolished in cells carrying SSA1 cysteine mutant alleles. Taken together, these findings demonstrate that Hsp70 acts as a sensor to induce the cytoprotective heat shock response in response to environmental or endogenously produced thiol-reactive molecules and can discriminate between two distinct environmental stressors.