Sandy D. Westerheide

Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1

Heat shock factor 1 (HSF1) is essential for protecting cells from protein-damaging stress associated with misfolded proteins and regulates the insulin-signaling pathway and aging. Here, we show that human HSF1 is inducibly acetylated at a critical residue that negatively regulates DNA binding activity. Activation of the deacetylase and longevity factor SIRT1 prolonged HSF1 binding to the heat shock promoter Hsp70 by maintaining HSF1 in a deacetylated, DNA–binding competent state. Conversely, down-regulation of SIRT1 accelerated the attenuation of the heat shock response (HSR) and release of HSF1 from its cognate promoter elements. These results provide a mechanistic basis for the requirement of HSF1 in the regulation of life span and establish a role for SIRT1 in protein homeostasis and the HSR.

Celastrols as inducers of the heat shock response and cytoprotection

Alterations in protein folding and the regulation of conformational states have become increasingly important to the functionality of key molecules in signaling, cell growth, and cell death. Molecular chaperones, because of their properties in protein quality control, afford conformational flexibility to proteins and serve to integrate stress-signaling events that influence aging and a range of diseases including cancer, cystic fibrosis, amyloidoses, and neurodegenerative diseases. We describe here characteristics of celastrol, a quinone methide triterpene and an active component from Chinese herbal medicine identified in a screen of bioactive small molecules that activates the human heat shock response. From a structure/function examination, the celastrol structure is remarkably specific and activates heat shock transcription factor 1 (HSF1) with kinetics similar to those of heat stress, as determined by the induction of HSF1 DNA binding, hyperphosphorylation of HSF1, and expression of chaperone genes. Celastrol can activate heat shock gene transcription synergistically with other stresses and exhibits cytoprotection against subsequent exposures to other forms of lethal cell stress. These results suggest that celastrols exhibit promise as a new class of pharmacologically active regulators of the heat shock response.

Triptolide, an Inhibitor of the Human Heat Shock Response That Enhances Stress-induced Cell Death *□

Molecular chaperones, inducible by heat shock and a variety of other stresses, have critical roles in protein homeostasis, balancing cell stress with adaptation, survival, and cell death mechanisms. In transformed cells and tumors, chaperones are frequently overexpressed, with constitutive activation of the heat shock transcription factor HSF1 implicated in tumor formation. Here, we describe the activity of triptolide, a diterpene triepoxide from the plant Triptergium wilfordii, as an inhibitor of the human heat shock response. Triptolide treatment of human tissue culture cells prevented the inducible expression of heat shock genes, shown by suppression of an HSP70 promoter-reporter construct and by suppression of endogenous HSP70 gene expression. Upon examining the steps in the HSF1 activation pathway, we found that triptolide abrogates the transactivation function of HSF1 without interfering in the early events of trimer formation, hyperphosphorylation, and DNA binding. The ability of triptolide to inhibit the heat shock response renders these cells sensitive to stress-induced cell death, which may be of great relevance to cancer treatments.

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 celastrols 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.

PTEN blocks TNF-induced NF-kappa B-dependent transcription by inhibiting the transactivation potential of the p65 subunit

PTEN is a lipid phosphatase responsible for down-regulating the phosphoinositide 3-kinase product phosphatidylinositol 3,4,5-triphosphate. Phosphatidylinositol 3,4,5-triphosphate is involved in the activation of the anti-apoptotic effector target, Akt. Although the Akt pathway has been implicated in regulating NF-κB activity, it is controversial as to whether Akt activates NF-κB predominantly through mechanisms that regulate nuclear translocation or transactivation potential. In this report, we utilized PTEN as a natural biological inhibitor of Akt activity to study the effects on tumor necrosis factor (TNF)-induced activation of NF-κB. We found that the reintroduction of PTEN into prostate cells inhibited TNF-stimulated NF-κB transcriptional activity. PTEN failed to block TNF-induced IKK activation, IκBα degradation, p105 processing, p65 (RelA) nuclear translocation, and DNA binding of NF-κB. However, PTEN inhibited NF-κB-dependent transcription by blocking the ability of TNF to stimulate the transactivation domain of the p65 subunit. PTEN also inhibited the transactivation potential of the cyclic AMP-response element-binding protein, but this was not observed for c-Jun. The transactivation potential of p65 following TNF stimulation could be rescued from PTEN-dependent repression by re-introducing expression constructs encoding activated forms of phosphoinositide 3-kinase, Akt, or Akt and IKK. The ability of PTEN to inhibit the TNF-induced transactivation function of p65 is important, because expression of PTEN blocked TNF-stimulated NF-κB-dependent gene expression, thus sensitizing cells to TNF-induced apoptosis. Maintenance of the PTEN tumor suppressor protein is therefore required to modulate Akt activity and to concomitantly control the transcriptional activity of the anti-apoptotic transcription factor NF-κB.

The Heat Shock Response and the Stress of Misfolded Proteins

Publisher Summary The heat shock response (HSR) is an inducible molecular response to a disruption of protein homeostasis that results in the elevated expression of cytoprotective genes that protect the proteome. The four general categories of environmental and physiological regulators of the HSR include: environmental stress, such as heat shock, amino acid analogs, drugs, oxidative stress, toxic chemicals, heavy metals, and pharmacologically active small molecules; cell growth and developmental conditions, including cell cycle, growth factors, development, differentiation, and activation by certain oncogenes; pathology and disease, such as neuroendocrine stress, tissue injury and repair, fever, inflammation, infection, ischemia and reperfusion, and cancer; and diseases of protein conformation including Huntingtons disease, Alzheimers disease, Parkinsons disease, and ALS. The HSR, through the elevated synthesis of molecular chaperones and proteases, responds rapidly and precisely to the intensity and duration of specific environmental and physiological stress signals to restore proteostasis and prevent further protein damage. Transient exposure to intermediate elevated temperatures or lower levels of chemical and environmental stress has cytoprotective effects against sustained, normally lethal, exposures to stress. HSFs are highly conserved and are required for normal cell growth and development in addition to their central importance in stress adaptation, survival, and disease. HSF1 as the principal stress activated factor binds to heat shock elements (HSEs) consisting of multiple contiguous inverted repeats of the pentamer sequence nGAAn located in the promoter regions of all heat shock responsive genes in eukaryotes.