Browsing by Subject "Stress response"

Heat Shock transcription Factor 1 (HSF1) has long been recognized as the master regulator and signal integrator in the eukaryotic proteotoxic stress response. Revealed by recent discoveries in cancer, the functions of HSF1 have extended far beyond its canonical role in protein folding, further encompassing critical functions in anti-apoptosis, invasion and metastasis, energy metabolism, DNA damage repair, and evasion of host immune surveillance. Meanwhile, both our understanding of the molecular basis of HSF1 regulation as well as available biochemical tools to investigate such details are lacking. Based on an in vitro ligand binding approach, the studies presented in this thesis were dedicated to the identification, validation, and characterization of a direct, first-in-class, small-molecule HSF1 inhibitor. The pharmacological inhibition of HSF1 occurs through small-molecule stimulation of nuclear, but not cytoplasmic HSF1 degradation, which attenuated prostate cancer cell proliferation, inhibited the HSF1 cancer gene signature and arrested tumor progression in multiple therapy-resistant animal models of prostate cancer. The identification of a direct small-molecule HSF1 inhibitor provides a unique pharmacological tool for future HSF1 research and serves as a significant proof-of-concept for pharmacologically targeting HSF1 for anti-cancer treatment approaches.

Stress tolerance and adaptability to dynamic environments are two things that make a microbial pathogen especially dangerous in the setting of a human infection. Cryptococcus neoformans, a ubiquitous pathogenic fungus, is able to sense, adapt, and tolerate the stressful environment of the human host in order to survive and cause disease. From the time this pathogen is inhaled into the lung to when it enters the central nervous system to cause life-threatening cryptococcal meningoencephalitis, C. neoformans activates numerous stress response signaling pathways to convert extracellular cues into adaptive cellular responses to ensure its survival in a new environment. Upon entering the human host, C. neoformans must overcome the stress of increased extracellular pH in order to survive. This organism is naturally found in environmental reservoirs with a pH of 5-6, but must adapt to a relatively alkaline pH pf 7.4 in niches of the human host such as the blood stream and interstitial alveolar space. Our work focuses on the ability for this fungal pathogen to modify both its cell wall and cell membrane using pH-response signaling pathways in order to thrive in an alkaline environment. Elucidating the mechanism of this pH response will not only help us understand the way this particular pathogen adapts to novel environments, but also reveal how we might manipulate certain components or processes in these adaptive signaling pathways to prevent and treat this invasive fungal infection. One example of a known external pH-sensing process in many model fungi and fungal pathogens is the Rim/Pal signal transduction pathway. Mutations in this pathway result in strains that are attenuated for survival at alkaline pH, and often for survival within the host due to the role for this pathway in cell wall remodeling and maintenance. We used an insertional mutagenesis screen to identify novel upstream components in the Rim pathway required for C. neoformans growth at host pH. We discovered altered alkaline pH growth in several strains with specific defects in plasma membrane composition and maintenance of phospholipid assembly. Among these, loss of function of the Cdc50 lipid flippase regulatory subunit affected the temporal dynamics of Rim pathway activation. Lipid flippase complexes, including Cdc50, are essential for maintaining the asymmetric distribution of phospholipids in the plasma membrane. We explored how Cdc50-mediated maintenance of lipid asymmetry affect membrane-bound pH-sensing proteins in the Rim pathway to facilitate signaling. Specifically, we demonstrated how the upstream Rim pathway activator and pH sensor, Rra1, uses its C-terminal tail to sense these alterations in lipid asymmetry and activate the downstream portion of the pathway. These results suggest both broadly applicable and phylum-specific molecular interactions that drive microbial environmental sensing involving the Rim alkaline response pathway. The ability for cells to internalize extracellular cues allows them to adapt to novel and stressful environments. The Rim pathway effectively converts the extracellular signal of increased pH into an adaptive cellular response allowing the pathogen to survive in its new environment. As previously mentioned, Rra1 is a plasma membrane protein responsible for sensing and internalizing the alkaline pH signal. We further identify the specific mechanisms of Rim pathway signaling through detailed studies of the activation of the Rra1 protein. Specifically, we observe that the Rra1 protein is internalized and recycled in a pH-dependent manner and that this further depends on specific residues on its C-terminal tail, clathrin-mediated endocytosis, and the integrity of the plasma membrane. These results continue to unravel the complex and intricate dynamics of membrane-mediated pH-sensing in a relevant human fungal pathogen. Observations from our genetic screen revealed that the C. neoformans sterol homeostasis pathway is required for growth at elevated pH. We find that an elevated pH is sufficient to induce activation of the sterol homeostasis pathway transcription factor, Sre1. This pH-mediated activation of the Sre1 transcription factor is linked to the biosynthesis of ergosterol, but is not dependent on Rim pathway signaling, indicating that these two pathways are responding to alkaline pH independently. Furthermore, we discover that C. neoformans is more susceptible to membrane-targeting antifungals under alkaline conditions, highlighting the impact of microenvironmental pH on the treatment of invasive fungal infections. Together, these findings further connect membrane integrity and composition with the fungal pH response. Rim-mediated modifications of both fungal cell wall components and membrane lipids combined with the ergosterol essentiality in the ability for fungal cells to grow in alkaline environments led us to explore the cell exterior in more detail. We include a comprehensive review of what is currently known in the field about the backbone structures of the cell wall: chitin and chitosan. A greater understanding of the complex layering that composes the structures connected to the plasma membrane will elucidate the barrier function these components provide in the collective response to pH stress. These studies revealing exploring the mechanisms of the alkaline pH response in a relevant human fungal pathogen will enhance our understanding of how these microorganisms tolerate and overcome the stressful host environment. Furthermore, the fact that these alkaline signaling pathways intimately involve the dynamics of the plasma membrane, further elucidate the general mechanisms by which cells respond to and internalize changes in extracellular environments using the exterior architecture of the cell.

Heat Shock Transcription Factor 1 (HSF1) is a critical regulator of transcription that facilitates cellular stress protection in response to protein misfolding, rapid cell proliferation, and other stressful conditions. Defective HSF1 regulation is observed in cellular and animal models of cancer, where hyperactive and dysregulated HSF1 supports cancer survival, and in neurodegenerative disease, where HSF1 function is compromised, further exacerbating protein misfolding. HSF1 is tightly regulated through intramolecular interactions, post-translational modifications, and protein-protein interactions; however, little is known about ho HSF1 regulation differs in response to stresses such as acute or chronic protein misfolding.

We identified one mechanism that contributes to the diminution of HSF1 in chronic protein misfolding in the context of Huntington’s Disease involving inappropriate interactions of HSF1 with CK2α’ and FBXW7 E3 ligase. We found these protein-protein interactions coordinate the abnormal phosphorylation-dependent degradation of HSF1. Importantly, inhibition of this aberrant HSF1 degradation attenuates the biochemical defects and protein misfolding in Huntington’s Disease. To further elucidate how HSF1-interacting proteins regulate HSF1 in acute and chronic stress, we carried out quantitative proteomics studies of the HSF1 interactome under control, acute heat shock, and in a cell model of Huntington’s Disease. We recapitulated many previously described interaction partners of HSF1 and identified several novel HSF1-interacting proteins that encompass a wide variety of cellular functions, including roles in DNA repair, mRNA processing, and regulation of RNA polymerase II. We further report on the interaction of HSF1 with CCCTC binding factor (CTCF), which modulates target gene activation and repression function of HSF1 by facilitating DNA binding at CTCF and HSF1 co-regulated loci. Given the role and elevated expression of both pro-inflammatory proteins and Tau in Huntington’s Disease, and their defective repression by HSF1, understanding the mechanisms of HSF1 repression is of great interest. The studies presented in this thesis expand our understanding of HSF1-mediated gene activation and repression, and the regulation of HSF1 via protein-protein interactions.