Activation Of Heat Shock Response Research Articles

Stress response mechanisms in protein misfolding diseases: Profiling a cellular model of Huntington's disease

Stress response pathways like the integrated stress response (ISR), the mitochondrial unfolded protein response (UPRmt) and the heat shock response (HSR) have emerged as part of the pathophysiology of neurodegenerative diseases, including Huntington's disease (HD) – a currently incurable disease caused by the production of mutant huntingtin (mut-Htt). Previous data from HD patients suggest that ISR is activated while UPRmt and HSR are impaired in HD. The study of these stress response pathways as potential therapeutic targets in HD requires cellular models that mimic the activation status found in HD patients of such pathways. PC12 cells with inducible expression of the N-terminal fragment of mut-Htt are among the most used cell lines to model HD, however the activation of stress responses remains unclear in this model. The goal of this study is to characterize the activation of ISR, UPRmt and HSR in this HD cell model and evaluate if it mimics the activation status found in HD patients. We show that PC12 HD cell model presents reduced levels of Hsp90 and mitochondrial chaperones, suggesting an impaired activation or function of HSR and UPRmt. This HD model also presents increased levels of phosphorylated eIF2α, the master regulator of the ISR, but overall similar levels of ATF4 and decreased levels of CHOP - transcription factors downstream to eIF2α - in comparison to control, suggesting an initial activation of ISR. These results show that this model mimics the ISR activation and the impaired UPRmt and HSR found in HD patients. This work suggests that the PC12 N-terminal HD model is suitable for studying the role of stress response pathways in the pathophysiology of HD and for exploratory studies investigating the therapeutic potential of drugs targeting stress responses.

Kainic Acid-Induced Excitotoxicity Leads to the Activation of Heat Shock Response.

Heat shock response (HSR) which is regulated by heat shock factor 1 (HSF1) is the most important mechanism and the major regulator that prevents protein aggregation in neurodegenerative diseases. Excitotoxicity, which is the accumulation of excess glutamate in synaptic cleft, is observed in age-dependent neurodegenerative diseases and also in stroke, epilepsy, and brain trauma. Only a few studies in the literature show the link between excitotoxicity and HSR. In this study, we aimed to show the molecular mechanism underlying this link. We applied heat shock (HS) treatment and induced excitotoxicity with kainic acid (KA) in neuroblastoma (SHSY-5Y) and glia (immortalized human astrocytes (IHA)) cells. We observed that, only in SHSY-5Y cells, heat shock preconditioning increases cell survival after KA treatment. GLT-1 mRNA expression is increased as a result of KA treatment and HS due to the elevation of HSF1 binding to GLT-1 promoter which was induced by HSF1 phosphorylation and sumolation in SHSY-5Y cells. Additionally, glutamine synthetase and glutaminase expressions are increased after HS preconditioning in SHSY-5Y cells indicating that HS activates glutamate metabolism modulators and accelerates the glutamate cycle. In glia cells, we did not observe the effect of HS preconditioning. In summary, heat shock preconditioning might be protective against excitotoxicity-related cell death and degeneration.

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy.

Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response.

The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) - rather than unfolded mature proteins - and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here, we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 - but not Sis1 - attenuates the response.

RHODIOLA ROSEA AND FERULIC ACID ACTIVATE EXPRESSION OF GENES RELATED TO AUTOPHAGY AND RESISTANCE TO HEAT SHOCK IN MICE OF DIFFERENT AGE

The aim of our study was testing whether R. rosea extract and ferulic acid activate expression of targets of FoxO, regulators of energy metabolism and autophagy in livers of young and old mice, and to what extent the effects of R. rosea extract and ferulic acid on the genes studied coincide. Methods. . C57BL/6J mice were reared at 22 ± 2 °С, 50-60% humidity, and 12/12 hour light/dark cycle. All groups were reared on a standard chow (4,8% fats, 21,8% protein, and 3,9% fibre). Experimental groups consumed water, supplemented with either sodium ferulate or R. rosea during 12 weeks prior sacrificing. The amounts of ferulate and R. rosea were adjusted to provide 4 mg of phenol-containing substances per 100 g weight, for a mouse, for 24 hours. We tested three-month-old (“young”) and twelve-month-old males (“old”). The levels of messenger ribonucleic acid (mRNA) were assessed using AriaMx real-time polymerase chain reaction (RT-PCR) instrument (Agilent). Ribonucleic acid was purified using the Monarch Miniprep kit (New England BioLabs (NEB), T2010), complementary deoxyribonucleic acid synthesis was performed using the ProtoScript II kit (NEB, E6560), and quantitative RT-PCR (qRT-PCR) was performed using the Luna Universal kit (NEB, E3003). The expression of genes ATG5 (an autophagy marker), HSPB8 (a small heat shock protein, an FoxO target), UCP2 (uncoupling protein 2, a senescence marker), CDKN2 (cell cycle regulator, a senescence marker), PDK2 and PDK4 (pyruvate dehydrogenase kinases 2 and 4, regulators of oxidative metabolism), and TFEB (transcription factor EB, a transcriptional regulator of autophagy) was evaluated. Results. Livers of young mice that consumed food supplemented with either sodium ferulate or R. rosea extract had 3.2-fold and 3.6-fold higher levels of mRNA of the small heat shock protein HspB8 than control mice, respectively. In old mice, the levels of mRNA for this protein were 3.3-fold higher in mice reared on the diet containing R. rosea extract as compared with the control. However, there was no significant difference between control mice and those that consumed ferulate-supplemented food. In young mice, ferulate and R. rosea extract induced synthesis of mRNA of PDK4 by 4.3 and 6.6 times from the control level, respectively. Ferulate and R. rosea extract also affected the levels of mRNA of ATG5 and PDK2 in the livers of old mice. The levels of PDK2 were 1.5-fold higher in the livers of mice that consumed ferulate-supplemented food than in control mice. Conclusions. Both, R. rosea extract and one of its active components – ferulic acid – promote increasing in the levels of mRNA for genes HSPB8 and PDK4, coding for small heat shock protein and pyruvate dehydrogenase kinase 4, respectively. In old mice, R. rosea promote expression of HSPB8, ATG5, PDK2, and PDK4. Thus, ferulic acid and R. rosea exert similar effects on gene expression by supposed activation of heat shock response and autophagy, and concomitant inhibition of mitochondrial metabolism via boosting expression of PDK2 and PDK4.

FUS ALS neurons activate major stress pathways and reduce translation as an early protective mechanism against neurodegeneration.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder causing progressive loss of motor neurons. Mutations in Fused in sarcoma (FUS) leading to its cytoplasmic mislocalization cause a subset of ALS. Under stress, mutant FUS localizes to stress granules (SGs)-cytoplasmic condensates composed of RNA and various proteins. Aberrant dynamics of SGs is linked to the pathology of ALS. Here, using motor neurons (MNs) derived from human induced pluripotent stem cells, we show that, in mutant FUS, MN dynamics of SGs is disturbed. Additionally, heat-shock response (HSR) and integrated stress response (ISR) involved in the regulation of SGs are upregulated in mutant MNs. HSR activation correlates with the amount of cytoplasmic FUS mislocalization. While inhibition of SG formation, translation, or ISR does not influence survival of FUS ALS neurons, proteotoxicity that cannot be compensated with the activation of stress pathways is the main driver of neurodegeneration in early FUS ALS.

Geranylgeranylacetone Ameliorates Beta-Amyloid Toxicity and Extends Lifespan via the Heat Shock Response in Caenorhabditis elegans.

Activation of a cytoprotective cellular pathway known as the heat shock response (HSR) is a promising strategy for the treatment of Alzheimer’s disease and other neurodegenerative diseases. Geranylgeranylacetone (GGA) is a commonly used anti-ulcer drug in Japan that has been shown to activate the HSR. Here, we establish C. elegans as a model system to investigate the effects of GGA. First, we show that GGA-mediated activation of the HSR is conserved in worms. Then, we show that GGA can ameliorate beta-amyloid toxicity in both muscle and neuronal worm Alzheimer’s disease models. Finally, we find that exposure to GGA is sufficient to extend the lifespan of wild-type worms. Significantly, the beneficial effects of GGA on both beta-amyloid toxicity and lifespan are dependent on HSR activation. Taken together, this research supports further development of GGA as a therapeutic for Alzheimer’s disease, provides evidence that HSR activation is a relevant therapeutic mechanism, and indicates that the beneficial effects of GGA are not limited to disease.

The CBP-1/p300 Lysine Acetyltransferase Regulates the Heat Shock Response in C. elegans.

The decline of proteostasis is a hallmark of aging that is, in part, affected by the dysregulation of the heat shock response (HSR), a highly conserved cellular response to proteotoxic stress in the cell. The heat shock transcription factor HSF-1 is well-studied as a key regulator of proteostasis, but mechanisms that could be used to modulate HSF-1 function to enhance proteostasis during aging are largely unknown. In this study, we examined lysine acetyltransferase regulation of the HSR and HSF-1 in C. elegans. We performed an RNA interference screen of lysine acetyltransferases and examined mRNA expression of the heat-shock inducible gene hsp-16.2, a widely used marker for HSR activation. From this screen, we identified one acetyltransferase, CBP-1, the C. elegans homolog of mammalian CREB-binding protein CBP/p300, as a negative regulator of the HSR. We found that while knockdown of CBP-1 decreases the overall lifespan of the worm, it also enhances heat shock protein production upon heat shock and increases thermotolerance of the worm in an HSF-1 dependent manner. Similarly, we examined a hallmark of HSF-1 activation, the formation of nuclear stress bodies (nSBs). In analyzing the recovery rate of nSBs, we found that knockdown of CBP-1 enhanced the recovery and resolution of nSBs after stress. Collectively, our studies demonstrate a role of CBP-1 as a negative regulator of HSF-1 activity and its physiological effects at the organismal level upon stress.

Withaferin A Induces Heat Shock Response and Ameliorates Disease Progression in a Mouse Model of Huntington's Disease.

Impairment of proteostasis network is one of the characteristic features of many age-related neurodegenerative disorders including autosomal dominantly inherited Huntington's disease (HD). In HD, N-terminal portion of mutant huntingtin protein containing expanded polyglutamine repeats accumulates as inclusion bodies and leads to progressive deterioration of various cellular functioning including proteostasis network. Here we report that Withaferin A (a small bioactive molecule derived from Indian medicinal plant, Withania somnifera) partially rescues defective proteostasis by activating heat shock response (HSR) and delays the disease progression in a HD mouse model. Exposure of Withaferin A activates HSF1 and induces the expression of HSP70 chaperones in an in vitro cell culture system and also suppresses mutant huntingtin aggregation in a cellular model of HD. Withaferin A treatment to HD mice considerably increased their lifespan as well as restored progressive motor behavioral deficits and declined body weight. Biochemical studies confirmed the activation of HSR and global decrease in mutant huntingtin aggregates load accompanied with improvement of striatal function in Withaferin A-treated HD mouse brain. Withaferin A-treated HD mice also exhibit significant decrease in inflammatory processes as evident from the decreased microglial activation. These results indicate immense potential of Withaferin A for the treatment of HD and related neurodegenerative disorders involving protein misfolding and aggregation.

Activation of heat shock response improves biomarkers of NAFLD in patients with metabolic diseases.

Nonalcoholic fatty liver disease (NAFLD) is often accompanied by metabolic disorders such as metabolic syndrome and type 2 diabetes (T2DM). Heat shock response (HSR) is one of the most important homeostatic abilities but is deteriorated by chronic metabolic insults. Heat shock (HS) with an appropriate mild electrical stimulation (MES) activates HSR and improves metabolic abnormalities including insulin resistance, hyperglycemia and inflammation in metabolic disorders. To analyze the effects of HS + MES treatment on NAFLD biomarkers, three cohorts including healthy men (two times/week, n = 10), patients with metabolic syndrome (four times/week, n = 40), and patients with T2DM (n = 100; four times/week (n = 40) and two, four, seven times/week (n = 20 each)) treated with HS + MES were retrospectively analyzed. The healthy subjects showed no significant alterations in NAFLD biomarkers after the treatment. In patients with metabolic syndrome, many of the NAFLD steatosis markers, including fatty liver index, NAFLD-liver fat score, liver/spleen ratio and hepatic steatosis index and NAFLD fibrosis marker, aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio, were improved upon the treatment. In patients with T2DM, all investigated NAFLD steatosis markers were improved and NAFLD fibrosis markers such as the AST/ALT ratio, fibrosis-4 index and NAFLD-fibrosis score were improved upon the treatment. Thus, HS + MES, a physical intervention, may become a novel treatment strategy for NAFLD as well as metabolic disorders.

Dexamethasone-induced activation of heat shock response ameliorates seizure susceptibility and neuroinflammation in mouse models of Lafora disease

Heat shock response (HSR) is a conserved cytoprotective pathway controlled by the master transcriptional regulator, the heat shock factor 1 (HSF1), that activates the expression of heat shock proteins (HSPs). HSPs, as chaperones, play essential roles in minimizing stress-induced damages and restoring proteostasis. Therefore, compromised HSR is thought to contribute to neurodegenerative disorders. Lafora disease (LD) is a fatal form of neurodegenerative disorder characterized by the accumulation of abnormal glycogen as Lafora bodies in neurons and other tissues. The symptoms of LD include progressive myoclonus epilepsy, dementia, and cognitive deficits. LD is caused by the defects in the gene coding laforin phosphatase or the malin ubiquitin ligase. Laforin and malin are known to work upstream of HSF1 and are essential for the activation of HSR. Herein, we show that mice deficient for laforin or malin show reduced levels of HSF1 and their targets in their brain tissues, suggesting compromised HSR; this could contribute to the neuropathology in LD. Intriguingly, treatment of LD animals with dexamethasone, a synthetic glucocorticoid analogue, partially restored the levels of HSF1 and its targets. Dexamethasone treatment was also able to ameliorate the neuroinflammation and susceptibility to induced seizures in the LD animals. However, dexamethasone treatment did not show a significant effect on Lafora bodies or autophagy defects. Taken together, the present study establishes a role for HSR in seizure susceptibility and neuroinflammation and dexamethasone as a potential antiepileptic agent, suitable for further studies in LD.

Subcellular localization of the J-protein Sis1 regulates the heat shock response.

Cells exposed to heat shock induce a conserved gene expression program, the heat shock response (HSR), encoding protein homeostasis (proteostasis) factors. Heat shock also triggers proteostasis factors to form subcellular quality control bodies, but the relationship between these spatial structures and the HSR is unclear. Here we show that localization of the J-protein Sis1, a cofactor for the chaperone Hsp70, controls HSR activation in yeast. Under nonstress conditions, Sis1 is concentrated in the nucleoplasm, where it promotes Hsp70 binding to the transcription factor Hsf1, repressing the HSR. Upon heat shock, Sis1 forms an interconnected network with other proteostasis factors that spans the nucleolus and the surface of the endoplasmic reticulum. We propose that localization of Sis1 to this network directs Hsp70 activity away from Hsf1 in the nucleoplasm, leaving Hsf1 free to induce the HSR. In this manner, Sis1 couples HSR activation to the spatial organization of the proteostasis network.

Blockade of HSP70 by VER-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma

Proteasome inhibitor bortezomib is one of the most effective drugs currently available for the treatment of multiple myeloma (MM). However, the intrinsic and acquired resistance to bortezomib can limit its effectiveness. The activation of heat shock response has been characterized as a potential resistance mechanism protecting MM cells from bortezomib-induced cell death. In this study, in response to bortezomib therapy, we discovered that HSP70 is one of the most substantially upregulated heat shock proteins. In order to further explore approaches to sensitizing bortezomib-based treatment for MM, we investigated whether targeting HSP70 using a specific inhibitor VER-155008 combined with bortezomib could overcome the acquired resistance in MM. We found that HSP70 inhibitor VER-155008 alone significantly decreased MM cell viability. Moreover, the combination of VER-155008 and bortezomib synergistically induced MM cell apoptosis markedly in vitro. Notably, the combined treatment was found to increase the cleavage of PARP, an early marker of chemotherapy-induced apoptosis. Importantly, the reduction of anti-apoptotic Bcl-2 family member Bcl-2, Bcl-xL, and Mcl-1 and the induction of pro-apoptotic Bcl-2 family member BH3-only protein NOXA and Bim were confirmed to be tightly associated with the synergism. Finally, the ER stress marker CHOP (CCAAT-enhancer binding protein homologous protein), which can cause transcriptional activation of genes involved in cell apoptosis, was markedly induced by both VER-155008 and bortezomib. Taken together, our finding of a strong synergistic interaction between VER-155008 and bortezomib may support for combination therapy in MM patients in the future.

Anesthetic pretreatment confers thermotolerance on Saccharomyces cerevisiae yeast

Saccharomyces cerevisiae yeast, when pretreated with elevated temperatures, undergo adaptive changes that promote survival after an otherwise lethal heat stress. The heat shock response, a cellular stress response variant, mediates these adaptive changes. Ethanol, a low-potency anesthetic, promotes thermotolerance possibly through heat shock response activation. Therefore, we hypothesized other anesthetic compounds, like ethanol, may invoke the heat shock response to promote thermotolerance. To test this hypothesis, we pretreated yeast with a series of non-volatile anesthetic and anesthetic-related compounds and quantified survival following lethal heat shock (52 °C for 5 min). Most compounds invoked thermoprotection and promoted survival with a potency proportional to hydrophobicity: tribromoethanol (5.6 mM, peak survival response), trichloroethanol (17.8 mM), dichloroethanol (100 mM), monochloroethanol (316 mM), trifluoroethanol (177.8 mM), ethanol (1 M), isopropanol (1 M), propofol (316 μM), and carbon tetrabromide (32 μM). Thermoprotection conferred by pretreatment with elevated temperatures was “left shifted” by anesthetic co-treatment from (in °C) 35.3 ± 0.1 to 32.2 ± 0.1 with trifluoroethanol (177.8 mM), to 31.2 ± 0.1 with trichloroethanol (17.8 mM), and to 29.1 ± 0.3 with tribromoethanol (5.6 mM). Yeast in postdiauxic shift growth phase, relative to mid-log, responded with greater heat shock survival; and media supplementation with tryptophan and leucine blocked thermoprotection, perhaps by reversing the amino acid starvation response. Our results suggest S. cerevisase may serve as a model organism for understanding anesthetic toxicity and anesthetic preconditioning, a process by which anesthetics promote tissue survival after hypoxic insult.

Dietary restriction and gonadal signaling differentially regulate post-development quality control functions in Caenorhabditis elegans.

Protein homeostasis is remodeled early in Caenorhabditis elegans adulthood, resulting in a sharp decline in folding capacity and reduced ability to cope with chronic and acute stress. Endocrine signals from the reproductive system can ameliorate this proteostatic collapse and reshape the quality control network. Given that environmental conditions, such as food availability, impact reproductive success, we asked whether conditions of dietary restriction (DR) can also reverse the decline in quality control function at the transition to adulthood, and if so, whether gonadal signaling and dietary signaling remodel the quality control network in a similar or different manner. For this, we employed the eat‐2 genetic model and bacterial deprivation protocol. We found that animals under DR maintained heat shock response activation and high protein folding capacity during adulthood. However, while gonadal signaling required DAF‐16, DR‐associated rescue of quality control functions required the antagonistic transcription factor, PQM‐1. Bioinformatic analyses supported a role for DAF‐16 in acute stress responses and a role for PQM‐1 in cellular maintenance and chronic stress. Comparing the stress activation and folding capacities of dietary‐ and gonadal‐signaling mutant animals confirmed this prediction and demonstrated that each differentially impacts cellular quality control capabilities. These data suggest that the functional mode of cellular quality control networks can be differentially remodeled, affecting an organism's ability to respond to acute and chronic stresses during adulthood.

Stable transgenic C9orf72 zebrafish model key aspects of the ALS/FTD phenotype and reveal novel pathological features

A hexanucleotide repeat expansion (HRE) within the chromosome 9 open reading frame 72 (C9orf72) gene is the most prevalent cause of amyotrophic lateral sclerosis/fronto-temporal dementia (ALS/FTD). Current evidence suggests HREs induce neurodegeneration through accumulation of RNA foci and/or dipeptide repeat proteins (DPR). C9orf72 patients are known to have transactive response DNA binding protein 43 kDa (TDP-43) proteinopathy, but whether there is further cross over between C9orf72 pathology and the pathology of other ALS sub-types has yet to be revealed.To address this, we generated and characterised two zebrafish lines expressing C9orf72 HREs. We also characterised pathology in human C9orf72-ALS cases. In addition, we utilised a reporter construct that expresses DsRed under the control of a heat shock promoter, to screen for potential therapeutic compounds.Both zebrafish lines showed accumulation of RNA foci and DPR. Our C9-ALS/FTD zebrafish model is the first to recapitulate the motor deficits, cognitive impairment, muscle atrophy, motor neuron loss and mortality in early adulthood observed in human C9orf72-ALS/FTD. Furthermore, we identified that in zebrafish, human cell lines and human post-mortem tissue, C9orf72 expansions activate the heat shock response (HSR). Additionally, HSR activation correlated with disease progression in our C9-ALS/FTD zebrafish model. Lastly, we identified that the compound ivermectin, as well as riluzole, reduced HSR activation in both C9-ALS/FTD and SOD1 zebrafish models.Thus, our C9-ALS/FTD zebrafish model is a stable transgenic model which recapitulates key features of human C9orf72-ALS/FTD, and represents a powerful drug-discovery tool.

Modulation of heat-shock response is associated with Di (2-ethylhexyl) phthalate (DEHP)-induced cardiotoxicity in quail (Coturnix japonica)

Di(2-ethylhexyl) phthalate (DEHP) is an omnipresent environmental pollutant with endocrine disrupting properties. As a plasticizer, DEHP can be leach from the plastic to transfer the external environment and thus enters the animal food chain, causing serious damage to the animal organs. The heat-shock response (HSR) comprising heat-shock protein (HSPs) and heat-shock transcription factor (HSFs) plays a pivotal role in various toxic stress conditions. For the sake of investigating the effects of DEHP exposure on cardiac toxicity and the regulation of HSR, male quail were fed the diet with 0, 250, 500 and 750 mg/kg DEHP by gavage administration for 45 days. Histopathological changes including cardiomyocyte swelling and muscle fiber dilatation were observed in the hearts exposed to DEHP. During the DEHP treatment, the mRNA expression of HSP60 and HSP70 were universally reduced, while the expression of other HSPs (HSP10, HSP25, HSP27, HSP40, HSP47, HSP90, HSP110) had different degrees of growth. In addition, the levels of HSF1, HSF2, and HSF3 were significantly increased. Given the facts above, DEHP exposure induced the toxic effects of quail heart. DEHP exposure did great harm to HSR via affecting the synthesis of HSFs to mediate the transcription of the HSPs. Ultimately, this study provided new evidence that DEHP-induced cardiotoxicity in quail was related to activation of HSR and playing a protective role.

Protein Homeostasis: A Key Target in Myeloma

Multiple myeloma is a disease of long-lived plasma cells that remains incurable in the majority of patients. However advances in the treatment have been made in the last 20 years resulting in significant improvement in the survival of patients with this disease. Interestingly almost all of these advances have been made through the targeting of pathways or molecules that are shared between normal and malignant plasma cells and are not due to the precision targeting of myeloma cell dependencies associated with the transformation process. Most notably targeting both specific and bulk protein homeostasis through the action of IMiDs and proteasome inhibitors have become the backbone for the treatment of newly diagnosed myeloma in the United States. We have focused on proteasome function and regulation to better understand why a macromolecular structure that is present and functions in every cell in the body is such an effective target for myeloma therapy. Our initial findings indicated that proteasome inhibition activated an endoplasmic reticulum stress response known as the unfolded protein response (UPR). The UPR is required for normal differentiation and maintenance of plasma cells, although our initial findings suggesting that the PERK arm of the response, which is not required for normal plasma cell maintenance was activated by proteasome inhibitors. We determined that the likely source of unfolded protein was immunoglobulin produced by myeloma cells and that the fate of immunoglobulin was a key determinant of sensitivity to proteasome inhibition. We have now focused on additional factors that determine the response to proteasome inhibition and consistent with previous findings have demonstrated that activation of heat shock response is a early protective event following proteasome inhibition. However we were unable to demonstrate that any single heat shock protein was sufficient to alter the response to proteasome inhibition. Rather it was the activation of the heat shock response itself and that protection was only efficiently blocked by inhibition of the transcriptional regulator of the response, HSF1. HSF1 is activated through post-translational modifications and proteasome inhibition results in phosphorylation of HSF1 at several sites, most notably S326. This can be blocked with the multi-kinase inhibitor TG02 that effectively blocks the proteasome inhibitor-induced heat shock response and sensitizes myeloma cells to proteasome inhibitor-induced cell death. Based on these findings, the combination of TG02 and carfilzomib are being tested clinically. The cellular response to proteasome inhibitors is also controlled through the regulation of proteasome assembly. We have recently demonstrated that 14-3-3ζ an anti-apoptotic phospho-serine binding protein, can limit proteasome assembly and activity through its binding to the PA28α subunit of the 11S regulator. This significantly lowers proteasome capacity and sensitizes cells to proteasome inhibition but not to other therapeutic agents. Finally in addition to regulation of bulk protein homeostasis, targeting of specific protein turnover has proven effective in myeloma treatment. IMiDs can alter the ability of the E3 ligase cereblon to recognize substrates for ubiquitination, resulting in the degradation of key B cell transcription factors IKZF1/3. These can regulate the plasma cell maintenance factor IRF4 as well as play a direct role in the regulation plasma cell superenhancers that control the expression of oncogenes translocated in myeloma. Another key mediator of myeloma cell survival, MCL1 is regulated via protein stability and can targeted in this manner. Direct and indirect targeting of MCL1 will be discussed. Disclosures Boise: Abbvie: Consultancy; Eli Lilly and Company: Research Funding.

Transcriptional Regulation of the Ambient Temperature Response by H2A.Z Nucleosomes and HSF1 Transcription Factors in Arabidopsis

Temperature influences the distribution, range, and phenology of plants. The key transcriptional activators of heat shock response in eukaryotes, the heat shock factors (HSFs), have undergone large-scale gene amplification in plants. While HSFs are central in heat stress responses, their role in the response to ambient temperature changes is less well understood. We show here that the warm ambient temperature transcriptome is dependent upon the HSFA1 clade of Arabidopsis HSFs, which cause a rapid and dynamic eviction of H2A.Z nucleosomes at target genes. A transcriptional cascade results in the activation of multiple downstream stress-responsive transcription factors, triggering large-scale changes to the transcriptome in response to elevated temperature. H2A.Z nucleosomes are enriched at temperature-responsive genes at non-inducible temperature, and thus likely confer inducibility of gene expression and higher responsive dynamics. We propose that the antagonistic effects of H2A.Z and HSF1 provide a mechanism to activate gene expression rapidly and precisely in response to temperature, while preventing leaky transcription in the absence of an activation signal.

The heat shock response and humoral immune response are mutually antagonistic in honey bees

The honey bee is of paramount importance to humans in both agricultural and ecological settings. Honey bee colonies have suffered from increased attrition in recent years, stemming from complex interacting stresses. Defining common cellular stress responses elicited by these stressors represents a key step in understanding potential synergies. The proteostasis network is a highly conserved network of cellular stress responses involved in maintaining the homeostasis of protein production and function. Here, we have characterized the Heat Shock Response (HSR), one branch of this network, and found that its core components are conserved. In addition, exposing bees to elevated temperatures normally encountered by honey bees during typical activities results in robust HSR induction with increased expression of specific heat shock proteins that was variable across tissues. Surprisingly, we found that heat shock represses multiple immune genes in the abdomen and additionally showed that wounding the cuticle of the abdomen results in decreased expression of multiple HSR genes in proximal and distal tissues. This mutually antagonistic relationship between the HSR and immune activation is unique among invertebrates studied to date and may promote understanding of potential synergistic effects of disparate stresses in this critical pollinator and social insects more broadly.