A Chlamydia trachomatis heat shock protein 60kDa (chsp60) exhibits increased expression in response to iron limitation. Genome sequencing revealed three genes encoding chsp60s. The objective of this study was to generate peptide antisera that would selectively recognize each chsp60. The DNA sequence for each C. trachomatis serovar E chsp60 was determined and compared with existing genome sequences. Predictive amino acid sequences were evaluated for peptides unique to each chsp60. Synthetic peptides were used to generate antisera; the resultant sera were purified by affinity chromatography and adsorbed to reduce cross-reactivity and increase monospecificity. Antisera were evaluated against each recombinant chsp60 protein by Western blotting. Reactivity against native chsp60s was visualized by transmission electron microscopy. Initial experiments indicate that expression of the second chsp60 (encoded by groEL_2) is increased during iron limitation. The production of chsp60 antibodies in human patients is associated with damaging sequelae in chlamydial genital and ocular infections.

Thermal adaptation in freshwater planktonic crustacean Daphnia pulex has been investigated by means of heatshock (35°C) experiments using individuals acclimated to different water temperatures (18, 24, and 28°C). Retention of mobility and survival during the heatshock experiments differed among clones and strongly depended on acclimation temperature: clones acclimated to substressful temperature (28°C) survived significantly longer than clones acclimated to lower temperatures. Both retention of mobility and survival correlated with geographic latitude of the clones' origin. There was, on the other hand, no significant correlation between clones' performance in heatshock experiments and hemoglobin content. Clones do not differ in the degree of the effect of acclimation on survival during heatshock but differ with respect to the effect of acclimation on mobility retention.

HspB2 is a small heat shock protein encoded on human chromosome 11. Less than 1000 base pairs away from HSPB2 and situated in a head-to-head orientation lies the gene encoding another small heat shock protein, CRYAB. Because they are uncommonly close to one another they share regulatory elements. In addition, they share protein homology as sHSPs, suggesting that they perhaps perform aimilar functions. SHSPs such as HspB2 and CryAB are traditionally thought to provide protective effects to cells in response to a variety of stress inducers. In response to stress they form complexes around misfolded proteins or proteins in danger of denaturation. HspB2 has been shown to exhibit protective effects during cellular stress and to localize to the Z-line of skeletal muscle. It has also been implicated in cardiac energetics, specifically in the production of ATP, however little is known about its molecular targets. Here I report the use of yeast two-hybrid screening to uncover the molecular targets of HspB2. I also detail the process by which the screens are performed as well as the verification steps, including co-precipitation experiments in mammalian cells. Through these studies we identify many novelbinding partners of HspB2, including CryAB as well as multiple muscle and mitochondrial proteins. Proteins discovered to bind to HspB2 include such proteins as actin and myosin, enzymes catalyzing various steps of glycolysis and the electron transport chain, as well as redox-, small heat shock protein-, kinase-, and electrolyte-related proteins, among others. Studies of the binding partners of HspB2 in cardiac tissue will provide important information clarifying the involvement of HspB2 in cardiac muscle maintenance and metabolism.

Understanding the effects of natural variation in environmental temperature on organisms and how those organisms evolve to live in different thermal environments is a central tenet of evolutionary physiology. Phenotypic differences among populations are the result of local adaptation, innate genetic differences between populations, and phenotypic plasticity, differential responses to the environment. Although not mutually exclusive, distinguishing between these paradigms can help illuminate species boundaries resulting from thermal limitations in physiology. For my dissertation, I examined geographic variation in measures of thermal physiology of the western fence lizard, Sceloporus occidentalis to understand the relative role of adaptation and acclimation in determining the thermal biology of populations of this species living in different thermal environments. To achieve this goal I measured three indices of physiological function; body temperature, thermal tolerance and heat shock protein (Hsp70) abundance, across geographic and seasonal variation in temperature. Furthermore, I examined variation in sprint speed performance before and after heat stress and its relationship to Hsp70 to determine if stress protein expression is a reliable indicator of whole organism physiological stress. I found that geographic location can have a major effect on thermal physiology and performance in S. occidentalis in that thermal tolerance, Hsp70, and sprint speed varied with site and season with warmer southern sites typically more heat adapted than cooler northern sites. I also found a trade off in thermal tolerance suggesting that specialization to temperature was occurring in these lizards. Finally, lizards with increased Hsp70 were typically slower after heat stress indicating that Hsp70 is a reliable indicator of organism stress. Despite these findings, there was no difference in body temperature among sites and seasonal patterns in thermal tolerance suggest that during certain times of the year plastic responses to temperature may mask adaptive differences. Here, I argue that temperature differences between sites has resulted in temperature adaptation at these sites, but that plastic responses to seasonal variation in temperature can become more important during certain times of the year. Although these relationships have been thoroughly studied in invertebrate organisms, further research should examine whether these patterns exist in other vertebrate ectotherm species.

Rhodococcus equi is an important pathogen in foals between one to six months of age and is a major cause of death in in these animals. In addition, R. equi has recently emerged as a significant opportunistic pathogen in immunosuppressed humans, especially those infected with HIV. Despite the ability of the organism to survive stressful growth conditions, for example, exposure to elevated temperature and oxygen radicals, the role of heat shock proteins in the pathogenesis of R. equi has not been well documented. In this project we developed and optimised methods to purify the heat shock protein DnaK from R. equi, using a combination of ion-exchange and affinity chromatography. The effectiveness of the purification protocols were assessed using SDS-PAGE and Western-blotting with anti-DnaK antibodies, and the enzymic activity of the purified DnaK was verified with an ATPase assay. ATPase assays were also used to investigate the roles of other heat shock proteins in enhancing the activity of DnaK.

Cardiovascular disease (CVD) is the leading cause of death globally, and tissue ischemia induces disorders including myocardial infarction, pulmonary arterial hypertension, and atherosclerosis. Ischemic conditions considerably alter cellular homeostasis as well as metabolism and can result in cardiovascular dysfunction. Therefore, there is a dire need to develop a novel treatment strategy for curing myocardial ischemia via manipulating cellular networking and metabolism. Furthermore, studies have shown that ischemic conditions induce cellular stress such that it modifies the expression of various cellular proteins and may even promote cell death. Cellular proteins must fold into a three-dimensional, native state to become functional. To ensure efficient folding and prevent aggregation, molecular chaperones assist with the folding/refolding process. Additionally, chaperones regulate cellular proteostasis through the ubiquitin-proteosome system (UPS) or autophagy. The heat-shock protein (HSP) is a molecular chaperone family upregulated during stress conditions to assist cells with proper protein folding, stability, and turnover. Specifically, the 70-kDa heat-shock protein (Hsp70) has numerous cytoprotective and immunomodulatory effects through its interaction with components of several cellular pathways. This interaction is dependent on nucleotide exchange factors (NEFs) to hold the open conformation of HSP70's nucleotide-binding domain (NBD). Proteins in the Bcl-2- associated athanogene (BAG) family are a group of co-chaperones that interact with the ATPase domain of HSP70 and help maintain homeostasis. The BAG family proteins (BAG1-6) share an evolutionarily conserved region at their C-termini (the BAG domain), and BAG5 specifically is unique in that it contains five of these domains. BAG5 plays a crucial role in maintaining cellular homeostasis and viability. This project explores the role of BAG5 as a co-chaperone and potential therapeutic tool to improve cardiomyocyte function under ischemic conditions.

Indiana University-Purdue University Indianapolis (IUPUI) / Heat Shock Protein 60/10 (HSP60/10, or GroEL/ES in bacteria) chaperonin systems play a critical role in protein homeostasis through facilitating proper folding of misfolded or partially folded polypeptides that are otherwise prone to aggregation. HSP60 chaperonins are highly conserved and essential in nearly all organisms studied thus far, making them a promising target for antibiotic development. Early high-throughput screens in the Johnson lab have identified five main scaffolds that, though hit-to-lead development, have been optimized for chaperonin inhibition and antimicrobial effects. While these initial studies have shown promising evidence to support the viability of a chaperonin-targeting antibiotic strategy, it was unclear whether the conservation of human HSP60 (48% identity to bacterial GroEL) would hinder this therapeutic strategy from advancing due to potential toxicity associated with off-target inhibition of the human homolog. Additionally, while chaperonin inhibition often correlated with cytotoxicity to the various pathogens studied, there was a clear need to investigate inhibitor mechanisms to 1) verify on-target effects, and 2) guide future development of more potent and selective chaperonin-targeting antibiotic candidates. Herein, we conduct a medium-throughput screening of known bioactive molecules, approved drugs, and natural products against both bacterial GroEL and human HSP60, demonstrating that most molecules exhibited low-to-no toxicity to human cells in culture, despite being near equipotent inhibitors of human HSP60 and E. coli GroEL in our refolding assays. Thus, sequence conservation between human HSP60 and bacterial GroELs does not necessarily predict toxicity in vivo. We then investigate inhibitory mechanisms of our most well-established inhibitor series, the phenylbenzoxazole (PBZ) series, identifying three binding sites whereby PBZ molecules modulate GroEL folding and ATPase functions in a site-specific manner, predominately through its ability to interact with its co-chaperone GroES. Finally, we demonstrate that two standard of care drugs for T. brucei infections, suramin and nifurtimox, may elicit their trypanocidal effects through inhibiting HSP60. Due to structural similarities, we then screened our N-acylhydrazone (NAH) and α,β-unsaturated ketone (ABK) series of HSP60 inhibitors against T. brucei, finding that they are highly potent and selective trypanocidal agents. Together, these studies further support HSP60 as a viable drug target for antibiotic development. / 2025-07-03

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