Characterization of glucocorticoid receptor (NR3C1) gene polymorphisms and a family-based association study with hypertension

Hypertension is usually defined as having values of systolic blood pressure ≥140 mmHg, diastolic blood pressure ≥90 mmHg. Hypertension is one of the main adverse effects of glucocorticoid on the cardiovascular system. Glucocorticoids are essential hormones, secreted from adrenal glands in circadian fashion. Glucocorticoid's effect on blood pressure is conveyed by the glucocorticoid receptor (NR3C1), an omnipresent nuclear transcription factor. Although polymorphisms in this gene have long been implicated to be a causal factor for cardiovascular diseases such as hypertension, no study has yet thoroughly interrogated the gene's polymorphisms for their effect on blood pressure levels. Therefore, I have first resequenced ∼30 kb of the gene, encompassing all exons, promoter regions, 5'/3' UTRs as well as at least 1.5 kb of the gene's flanking regions from 114 chromosome 5 monosomic cell lines, comprised of three major American ethnic groups—European American, African American and Mexican American. I observed 115 polymorphisms and 14 common molecularly phased haplotypes. A subset of markers was chosen for genotyping study populations of GENOA (Genetic Epidemiology Network of Atherosclerosis; 1022 non-Hispanic whites, 1228 African Americans and 954 Mexican Americans). Since these study populations include sibships, the family-based association test was performed on 4 blood pressure-related quantitative variables—pulse, systolic blood pressure, diastolic blood pressure and mean arterial pressure. Using these analyses, multiple correlated SNPs are significantly protective against high systolic blood pressure in non-Hispanic whites, which includes rsb198, a SNP formerly associated with beneficial body compositions. Haplotype association analysis also supports this finding and all p-values remained significant after permutation tests. I therefore conclude that multiple correlated SNPs on the gene may confer protection against high blood pressure in non-Hispanic whites. ^

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.

Hsp110 chaperones control client fate determination in the hsp70-Hsp90 chaperone system.

Heat shock protein 70 (Hsp70) plays a central role in protein homeostasis and quality control in conjunction with other chaperone machines, including Hsp90. The Hsp110 chaperone Sse1 promotes Hsp90 activity in yeast, and functions as a nucleotide exchange factor (NEF) for cytosolic Hsp70, but the precise roles Sse1 plays in client maturation through the Hsp70-Hsp90 chaperone system are not fully understood. We find that upon pharmacological inhibition of Hsp90, a model protein kinase, Ste11DeltaN, is rapidly degraded, whereas heterologously expressed glucocorticoid receptor (GR) remains stable. Hsp70 binding and nucleotide exchange by Sse1 was required for GR maturation and signaling through endogenous Ste11, as well as to promote Ste11DeltaN degradation. Overexpression of another functional NEF partially compensated for loss of Sse1, whereas the paralog Sse2 fully restored GR maturation and Ste11DeltaN degradation. Sse1 was required for ubiquitinylation of Ste11DeltaN upon Hsp90 inhibition, providing a mechanistic explanation for its role in substrate degradation. Sse1/2 copurified with Hsp70 and other proteins comprising the "early-stage" Hsp90 complex, and was absent from "late-stage" Hsp90 complexes characterized by the presence of Sba1/p23. These findings support a model in which Hsp110 chaperones contribute significantly to the decision made by Hsp70 to fold or degrade a client protein.

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.