ArticleLiterature Review

The Heat-Shock Response

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... The sum of these molecular changes underlies temperature response on the cellular level, whereby complex processes such as gene expression, cytoskeleton dynamics, and metabolism are affected on the longer timescale, resulting in manifold and wide-ranging changes across the cell. Besides the classical temperature shock response (Lindquist, 1986;Fujita, 1999), the mechanisms and functional relevance of these processes remain poorly understood. ...
... Live labeling with a fluorescent plasma membrane presynaptic marker Cholera Toxin B subunit showed no obvious difference in membrane organization, suggesting that the effects of cooling were likely not associated with changes in plasma membrane structure (Supplementary Figure 3B). To test whether the synaptic remodeling of inhibitory proteins required protein translation as per canonical temperature shock response (Lindquist, 1986;Fujita, 1999), we assessed the effect of cooling on Geph in the presence of translation elongation blocker Anisomycin (Aniso). Strikingly, this treatment did not affect the effect of cooling (Figures 3G,H), suggesting that the temperature-dependent plasticity of inhibitory synapses did not require translation. ...
... Our results provide a novel mechanistic insight into the mechanisms of temperature-dependent synaptic plasticity. Remarkably, we find that cooling-induced reorganization of inhibitory PSD can proceed in the absence of protein biosynthesis, suggesting that this form of synaptic plasticity is likely uncoupled from the classical temperature shock response (Lindquist, 1986;Fujita, 1999;Peretti et al., 2015). Instead, our results highlight the importance of temperaturedependent cytoskeletal dynamics (Li and Moore, 2020;Kubota et al., 2021), which is evidenced by the hypothermia-induced actin redistribution. ...
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The function of the central nervous system (CNS) is strongly affected by temperature. However, the underlying processes remain poorly understood. Here, we show that hypothermia and hyperthermia trigger bidirectional re-organization of presynaptic architecture in hippocampal neurons, resulting in synaptic strengthening, and weakening, respectively. Furthermore, hypothermia remodels inhibitory postsynaptic scaffold into enlarged, sparse synapses enriched in GABAA receptors. This process does not require protein translation, and instead is regulated by actin dynamics. Induction of hypothermia in vivo enhances inhibitory synapses in the hippocampus, but not in the cortex. This is confirmed by the proteomic analysis of cortical synapses, which reveals few temperature-dependent changes in synaptic content. Our results reveal a region-specific form of environmental synaptic plasticity with a mechanism distinct from the classic temperature shock response, which may underlie functional response of CNS to temperature.
... , 1998), density, starvation, poison, ultraviolet-C, and diapause (Sosalegowda et al., 2010;Tedeschiab et al., 2015;Zhang et al., 2015;Tedeschi et al., 2016). HSP70 and HSP90 are highly conserved in all eukaryotes and prokaryotes and consist of two highly conserved domains: an N-terminal ATP-binding domain and a C-terminal substrate-binding domain (Lindquist. 1986;Taipale et al., 2010). These proteins generally serve in regulating the adaptions of insects to adverse environments and serve as a predominant selfprotection mechanism (Chen et al., 2015a;Guo and Feng, 2018). The heat shock protein 70 family includes stressinducible genes (HSP70s) and constitutively expressed members or heat shock cogna ...
... The well-known mechanism used to cope with extreme temperatures is the expression of stress-inducible HSPs (Feder and Hofmann 1999;Sørensen et al., 2003;King and MacRae, 2015;Ma et al., 2021). The transcription of genes (mRNAs) encoding inducible heat shock proteins (in the response) appears to be temperature-sensitive in insects (Lindquist, 1986;Theodorakis and Morimoto, 1987;Morimoto, 1993;Sistonen et al., 1994;Prahlad and Morimoto, 2009;Lewis et al., 2016;Jin et al., 2020;Tian et al., 2020). However, the HSP expression of thermal tolerance or acclimation of O. furnacalis and the molecular mechanisms in its physiology, are poorly understood. ...
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Acclimation to abiotic stress plays a critical role in insect adaption and evolution, particularly during extreme climate events. Heat shock proteins (HSPs) are evolutionarily conserved molecular chaperones caused by abiotic and biotic stressors. Understanding the relationship between thermal acclimation and the expression of specific HSPs is essential for addressing the functions of HSP families. This study investigated this issue using the Asian corn borer Ostrinia furnacalis , one of the most important corn pests in China. The transcription of HSP genes was induced in larvae exposed to 33°C. Thereafter, the larvae were exposed to 43°C, for 2 h, and then allowed to recover at 27 C for 0, 0.5, 1, 2, 4, 6, and 8 h. At the recovery times 0.5–4 h, most population tolerates less around 1–3 h than without recovery (at 0 h) suffering continuous heat stress (43 C). There is no difference in the heat tolerance at 6 h recovery, with similar transcriptional levels of HSPs as the control. However, a significant thermal tolerance was observed after 8 h of the recovery time, with a higher level of HSP70 . In addition, the transcription of HSP60 and HSC70 (heat shock cognate protein 70) genes did not show a significant effect. HSP70 or HSP90 significantly upregulated within 1–2 h sustained heat stress (43 C) but declined at 6 h. Our findings revealed extreme thermal stress induced quick onset of HSP70 or HSP90 transcription. It could be interpreted as an adaptation to the drastic and rapid temperature variation. The thermal tolerance of larvae is significantly enhanced after 6 h of recovery and possibly regulated by HSP70 .
... The stress response is critical to the brain's ability to endure the challenges of age and disease, and m 6 A modulation of transcripts is associated with a range of stress responses in mammalian cells. Molecular chaperones crucial to the heat shock response are instrumental in understanding neurological disease, and preconditioning heat stress has been noted to suppress disease toxicity and increase longevity 3,41,42 . Therefore, we examined if there were changes in m 6 A upon the brain heat shock (HS) response in vivo. ...
... Given these data showing that the brain has a unique m 6 A response to HS, we investigated mRNA and protein changes of selected molecular chaperones known to increase with stress. Whole fly and whole head tissue undergo a typical Drosophila HS response 41 , with rapid upregulation of Hsp70 protein by 30 min of HS that persists through a 6 h recovery ( Supplementary Fig. 2a). However, the brain (dissected from the head capsule and eyes) showed a delayed upregulation of Hsp70 protein compared to whole fly and whole head (Supplementary Fig. 2a). ...
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N6-methyladenosine (m6A), the most prevalent internal modification on eukaryotic mRNA, plays an essential role in various stress responses. The brain is uniquely vulnerable to cellular stress, thus defining how m6A sculpts the brain’s susceptibility may provide insight to brain aging and disease-related stress. Here we investigate the impact of m6A mRNA methylation in the adult Drosophila brain with stress. We show that m6A is enriched in the adult brain and increases with heat stress. Through m6A-immunoprecipitation sequencing, we show 5′UTR Mettl3-dependent m6A is enriched in transcripts of neuronal processes and signaling pathways that increase upon stress. Mettl3 knockdown results in increased levels of m6A targets and confers resilience to stress. We find loss of Mettl3 results in decreased levels of nuclear m6A reader Ythdc1, and knockdown of Ythdc1 also leads to stress resilience. Overall, our data suggest that m6A modification in Drosophila dampens the brain’s biological response to stress. The brain is vulnerable to stress and disease, with much work focused on defining mechanisms that impact the brain’s resilience. Here the author’s reveal in Drosophila that m6A epitranscriptomic modification of RNA dampens the brain’s capacity to mitigate stress by regulating RNA stability and translation.
... Heat shock proteins (HSPs) are multi-family proteins that act as chaperones in nature and play a key role in the survival mechanism of organisms under various stresses [81]. They exist in five families based on their molecular weight, namely HSP100, HSP90, HSP70, HSP60/40, and HSP20 [82]. ...
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In the present era of climate change and global warming, high temperatures have increased considerably, posing a threat to plant life. Heat stress affects the biochemistry, physiology and molecular makeup of the plant by altering the key processes, i.e., photosynthesis, respiration and reproduction which reduces its growth and development. There is a dire need to manage this problem sustainably for plant conservation as well as the food security of the human population. Use of phytohormones to induce thermotolerance in plants can be a sustainable way to fight the adversities of heat stress. Phytohormone-induced thermotolerance proves to be a compelling approach to sustainably relieve the damaging effects of heat stress on plants. Salicylic acid (SA) is an essential molecule in biotic and abiotic defense response signal transduction pathways. When supplied externally, it imparts heat stress tolerance to the plants by different means, viz., increased Heat Shock Proteins (HSP) production, Reactive oxygen species (ROS) scavenging, protection of the reproductive system and enhancing photosynthetic efficiency. The effect of SA on plants is highly dependent on the concentration applied, plant species, plant age, type of tissues treated, and duration of the treatment. The present review paper summarizes the mechanism of thermotolerance induced by salicylic acid in plants under heat stress conditions. It includes the regulatory effects of SA on heat shock proteins, antioxidant metabolism, and maintenance of Ca 2+ homeostasis under heat stress. This review combines the studies conducted to elucidate the role of SA in the modulation of different mechanisms which lead to heat stress tolerance in plants. It discusses the mechanism of SA in protecting the photosynthetic machinery and reproductive system during high-temperature stress.
... Furthermore, stressors can interact synergistically, potentially resulting in larger effects than individual stressors alone. Inferring the effects of stress on fitness requires a broad array of assays, ranging from molecular assays, to behavioral studies, to an examination of species range shifts (e.g., the heat shock response [HSR] (Lindquist 1986); diel vertical migrations (e.g., Müller et al. 2018); shifts in species distributions towards higher elevations (e.g., Freitas et al. 2016;reviewed in Parmesan 2006;Pinsky et al. 2020;Walther et al. 2002)). Changes in gene expression, however, represent an early, immediate, measurable response that can be assayed in a laboratory environment under controlled conditions, and as such can be a useful indicator of the initial, direct impact of one or multiple stressors on large scale, long-term outcomes like fitness and distribution. ...
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Understanding the short- and long-term consequences of climate change is a major challenge in biology. For aquatic organisms, temperature changes and drought can lead to thermal stress and habitat loss, both of which can ultimately lead to higher mutation rates. Here, we examine the effect of high temperature and mutation accumulation on gene expression at two loci from the heat shock protein (HSP) gene family, HSP60 and HSP90. HSPs have been posited to serve as 'mutational capacitors' given their role as molecular chaperones involved in protein folding and degradation, thus buffering against a wide range of cellular stress and destabilization. We assayed changes in HSP expression across 5 genotypes of Daphnia magna, a sentinel species in ecology and environmental biology, with and without acute exposure to thermal stress and accumulated mutations. Across genotypes, HSP expression increased ~ 6× in response to heat and ~ 4× with mutation accumulation, individually. Both factors simultaneously (lineages with high mutation loads exposed to high heat) increased gene expression ~ 23×-much more than that predicted by an additive model. Our results corroborate suggestions that HSPs can buffer against not only the effects of heat, but also mutations-a combination of factors both likely to increase in a warming world. Supplementary information: The online version contains supplementary material available at 10.1007/s10682-022-10209-1.
... It's known that the psycho-emotional condition and behavioral reactions of humans are fundamentally changing depending on the temperature. For example, under the conditions of a high temperature the aggressive behavior of humans significantly changes, as well as the number of murder and suicide cases increases [1][2][3]. Due to it to produce a thermal energy as a result of the formation of adaptation syndrome, the specific thermoregulation mechanisms have been formed in humans and plants, among which the synthesis of heat-shock proteins in stress conditions have a special interest [4][5][6][7][8][9][10]. ...
... 5 Although under extreme conditions, the heat shock transcription factors (HSFs) are activated in response to stresses, leading the transcription of abundant HSPs to buffer the stresses. 7,8 And based on the primary molecular chaperon function, they also participate in multiple processes in eukaryotic cells, and malfunction of HSPs has reported to related with many diseases. In this review, we discuss the current understanding about the structures features and the chaperon functions of the major HSPs, and the regulation of HSPs by their cochaperons, describe the HSP network in proteostasis and how they response to the stresses. ...
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The heat shock proteins (HSPs) are ubiquitous and conserved protein families in both prokaryotic and eukaryotic organisms, and they maintain cellular proteostasis and protect cells from stresses. HSP protein families are classified based on their molecular weights, mainly including large HSPs, HSP90, HSP70, HSP60, HSP40, and small HSPs. They function as molecular chaperons in cells and work as an integrated network, participating in the folding of newly synthesized polypeptides, refolding metastable proteins, protein complex assembly, dissociating protein aggregate dissociation, and the degradation of misfolded proteins. In addition to their chaperone functions, they also play important roles in cell signaling transduction, cell cycle, and apoptosis regulation. Therefore, malfunction of HSPs is related with many diseases, including cancers, neurodegeneration, and other diseases. In this review, we describe the current understandings about the molecular mechanisms of the major HSP families including HSP90/HSP70/HSP60/HSP110 and small HSPs, how the HSPs keep the protein proteostasis and response to stresses, and we also discuss their roles in diseases and the recent exploration of HSP related therapy and diagnosis to modulate diseases. These research advances offer new prospects of HSPs as potential targets for therapeutic intervention. Heat shock proteins (HSPs) are in charge of proteostasis and buffer the stresses. Malfunction of HSPs lead to many diseases. In this review, we discuss the current understanding about the functions and structures of the major HSP families, the HSP network in proteostasis and their roles in diseases, and the prospects of these proteins as potential targets for therapeutic intervention.
... The heat shock response is a highly conservative defense adaptation response of the body, which is characterized by changes in gene expression in response to heat stress [30]. HSF1 is closely related to the HIV-1 life cycle. ...
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At present, the barrier to HIV-1 functional cure is the persistence of HIV-1 reservoirs. The “shock (reversing latency) and kill (antiretroviral therapy)” strategy sheds light on reducing or eliminating the latent reservoir of HIV-1. However, the current limits of latency-reversing agents (LRAs) are their toxicity or side effects, which limit their practicability pharmacologically and immunologically. Our previous research found that HSF1 is a key transcriptional regulatory factor in the reversion of HIV-1 latency. We then constructed the in vitro HSF1-knockout (HSF1-KO) HIV-1 latency models and found that HSF1 depletion inhibited the reactivation ability of LRAs including salubrinal, carfizomib, bortezomib, PR-957 and resveratrol, respectively. Furthermore, bortezomib/carfizomib treatment induced the increase of heat shock elements (HSEs) activity after HSF1-KO, suggesting that HSEs participated in reversing the latent HIV-1. Subsequent investigation showed that latent HIV-1-reversal by H2O2-induced DNA damage was inhibited by PARP1 inhibitors, while PARP1 was unable to down-regulate HSF1-depleted HSE activity, indicating that PARP1 could serve as a replaceable protein for HSF1 in HIV-1 latent cells. In summary, we succeeded in finding the mechanisms by which HSF1 reactivates the latent HIV-1, which also provides a theoretical basis for the further development of LRAs that specifically target HSF1.
... Israel Meteorological Service temperature data near A. hierochuntica populations during their growing season showed diurnal minimum : maximum night : day temperatures of c. 25°C and c. 40°C, respectively (Fig. S2). Thus, to simulate an ecologically relevant scenario with heat treatments that A. thaliana plants could also survive (Hayes et al., 2021), plants were exposed to similar three consecutive daily heat waves covering the early heat response and acquired heat tolerance phases (Lindquist, 1986;Hong & Vierling, 2000), with day : night temperatures of 40°C : 25°C followed by 2 d recovery at 23°C (Fig. 2a). To minimize shocks, temperatures were gradually ramped up and down at sunrise and sunset, respectively (Methods S1). ...
Article
Plant adaptation to a desert environment and its endemic heat stress is poorly understood at the molecular level. The naturally heat‐tolerant Brassicaceae species Anastatica hierochuntica is an ideal extremophyte model to identify genetic adaptations that have evolved to allow plants to tolerate heat stress and thrive in deserts. We generated an A. hierochuntica reference transcriptome and identified extremophyte adaptations by comparing Arabidopsis thaliana and A. hierochuntica transcriptome responses to heat, and detecting positively selected genes in A. hierochuntica. The two species exhibit similar transcriptome adjustment in response to heat and the A. hierochuntica transcriptome does not exist in a constitutive heat ‘stress‐ready’ state. Furthermore, the A. hierochuntica global transcriptome as well as heat‐responsive orthologs, display a lower basal and higher heat‐induced expression than in A. thaliana. Genes positively selected in multiple extremophytes are associated with stomatal opening, nutrient acquisition, and UV‐B induced DNA repair while those unique to A. hierochuntica are consistent with its photoperiod‐insensitive, early‐flowering phenotype. We suggest that evolution of a flexible transcriptome confers the ability to quickly react to extreme diurnal temperature fluctuations characteristic of a desert environment while positive selection of genes involved in stress tolerance and early flowering could facilitate an opportunistic desert lifestyle.
... The HSF1mediated activation of promoters with HSEs induces expression of heat shock proteins (HSPs) (Yamamoto, Takemori, Sakurai, Sugiyama, & Sakurai, 2009). The HSPs that can act as chaperones and chaperonins to help protein misfolding repair are expressed under heat stress (Lindquist, 1986). At first, the HSPs try to repair the aberrant proteins by refolding the proteins in a correct confirmation. ...
... Other powerful methods of restricting expression to single or fewer cells include the recombinase-based systems for stochastic labeling (Lis et al. 1983;Golic and Lindquist 1989;Xu and Rubin 1993;Lee and Luo 1999;Hadjieconomou et al. 2011;Nern et al. 2015;Isaacman-Beck et al. 2020), temperature sensitive mutations of Gal80, the repressor of GAL4 (Nogi et al. 1977;Lee and Luo 2001;McGuire et al. 2003), and use of transcription factors modified to drive transcription in the presence of an ingestible drug (McGuire et al. 2004). In addition to providing temporal and spatial control, however, these methods either involve increase in temperature that unleashes a stress response in all cells of the organism (Lindquist 1986), or addition of drugs with potential off-target effects, both of which may affect experimental outcomes. ...
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The ability to drive expression of exogenous genes in different tissues and cell types, under control of specific enhancers, has been crucial for discovery in biology. While many enhancers drive expression broadly, several genetic tools were developed to obtain access to isolated cell types. Studies of spatially organized neuropiles in the central nervous system of fruit flies have raised the need for a system that targets subsets of cells within a single neuronal type, a feat currently dependent on stochastic flip-out methods. To access the same cells within a given expression pattern consistently across fruit flies, we developed the light-gated expression system LOV-LexA. We combined the bacterial LexA transcription factor with the plant-derived light, oxygen or voltage photosensitive domain and a fluorescent protein. Exposure to blue light uncages a nuclear localizing signal in the C-terminal of the LOV domain, and leads to translocation of LOV-LexA to the nucleus, with subsequent initiation of transcription. LOV-LexA enables spatial and temporal control of expression of transgenes under LexAop sequences in larval fat body as well as pupal and adult neurons with blue light. The LOV-LexA tool is ready to use with GAL4 and Split-GAL4 drivers in its current form, and constitutes another layer of intersectional genetics, that provides light-controlled genetic access to specific cells across flies.
... Molecular chaperones, heat shock protein (HSPs), assist protein refolding [50]. According to previous reports, distinct reorganization of LRs is required to generate and transmit stress signals for stimulating HSP genes, thereby upregulating HSP expression [51]. ...
Article
With the rapid aging in the global population, delay of aging has become a hot research topic. Lipid rafts (LRs) are microdomains in the plasma membrane that contain sphingolipids and cholesterol. Emerging evidence indicates an interesting interplay between LRs and aging. LRs and their components are altered with aging. Further, the aging process is strongly influenced by LRs. In recent years, LRs and their component signaling molecules have been recognized to affect aging by interfering with its hallmarks. Therefore, targeting LRs is a promising strategy to delay aging.
... They probably represent a nonspecific cellular response to stress, as they can be affected by a wide number of stressors, whereas others may be specific to certain types of stress. Changes in gene expression and activity of expressed proteins are modified by nonlethal heat shock (Lindquist, 1986), resulting in a cellular response. This response involves a posterior increase in thermotolerance (such as the ability to adapt to subsequent, more severe heat stresses), associated with increased expression of HSPs. ...
Article
As the expectation of the influence of climate change scenarios increases, the effect of heat stress on small ruminants is significant due to their importance for smallholders in stressful environments. This article reviews the physical, physiological, metabolic, genes (N=126) and genetic pathways that affect heat stress response in sheep and goats. Molecular functions (N=304) linked to cytokine, growth factor, hormone and ATPase activity, as well as binding proteins, ATP, DNA, metal ions were prevalent in these analyses. As for biological processes (N=1803), gene expression and regulation, cell differentiation, apoptotic processes, cytokine pathways and inflammatory responses are important. Biological processes were centred on regulation, positive/negative, cell, signals, processes, proteins and pathways, while molecular functions included binding, DNA/RNA, protein, factors and activity. This review will contribute to have a better understanding of the complex animal´s response to heat stress and it should support scientific groups to delineate new studies as well to stimulate the generation of more data towards the development of strategies and methods to reach more sustainable animal production systems.
... Heat shock proteins are multifamily conserved proteins widely known to get produced by cells under stressful conditions. They can be produced during viral or bacterial infections, hypothermia, and cancer to generate a biological response known as heat shock response (HSR) (Lindquist, 1986). This heat shock response is primarily controlled by heat shock inducing factors (HSFs) such as HSF1, HSF2, HSF3, HSF4 and HSFY (Fujimoto & Nakai, 2010). ...
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Prostate cancer is a leading form of cancer among men of all ages worldwide. The androgen receptor pathway plays an important role in the development and functioning of this type of cancer. The pathway basically revolves around the functioning of androgens like testosterone or dihydrotestosterone (DHT) along with androgen receptors (ARs) to activate the special DNA binding sites or the androgen receptor elements (AREs) present in the nucleus. It leads to the activation of specific target genes such as ubiquitin conjugating enzyme E2 C (UBE2C), transmembrane protease, serine 2 : erythroblast transformation-specific related gene (TMPRSS2:ERG), glutathione S-transferase P (GSTP1), protein specific antigen (PSA), prostate cancer gene 3 (PCA3) and alpha-methylacyl CoA racemase (AMACR). These target genes are responsible for prostate carcinogenesis and progression or are an indicator of the same. The blocking of AR sites by AR inhibitors such as enzalutamide or by flutamide does not let the usual binding of androgens to occur and is one of the most commonly used effective methods to control prostate cancer. Though some of these AR inhibitors show certain side effects, therefore, the usage of common dietary compounds like resveratrol (Res) are being proposed as good therapeutic alternatives. This paper discusses another component involved in the AR pathway that is the Heat Shock Protein (HSP) and its types. The interaction of the HSP to the AR renders the AR inactive and helps maintain the AR in the correct spatial conformation in order to bind correctly to the androgens. The use of certain Heat Shock Protein (HSP) inhibitors such as VER155008 and Onalespib that bind to various HSPs disrupts the functioning of HSPs leading to an inhibitory effect on the AR expression. Hence, the AR pathway and its components are excellent therapeutic targets to control prostate cancer.
... The heat shock response is a highly conserved response of a cell to challenging environmental stress conditions [1], and the conserved set of proteins termed heat shock proteins (HSPs) is critical to the maintenance of cellular integrity under stress conditions. HSPs are primarily responsible for sustaining cellular protein homeostasis via their chaperone activity, by aiding the assembly and folding of proteins, and by inducing their degradation after irreversible damage [2]. ...
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Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone.
... The HSPs were first observed in Drosophila melanogaster in the early 1960s, as revealed by a "puffing" of genes in the chromosome of recovering cells [66]. The "puffing" has been shown to activate genes encoding the HSPs, which function as molecular chaperones [67]. The HSF family consists of four members in mammals, including HSF1, HSF2, HSF3, and HSF4. ...
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Thermal stress due to extreme changes in the thermal environment is a critical issue in cattle production. Many previous findings have shown a decrease in feed intake, milk yield, growth rate, and reproductive efficiency of the cattle when subjected to thermal stress. Therefore, selecting thermo-tolerant animals is the primary goal of the efficiency of breeding programs to reduce those adverse impacts. The recent advances in molecular genetics have provided significant breeding advantages that allow the identification of molecular markers in both beef and dairy cattle breeding, including marker-assisted selection (MAS) as a tool in selecting superior thermo-tolerant animals. Single-nucleotide polymorphisms (SNPs), which can be detected by DNA sequencing, are desirable DNA markers for MAS due to their abundance in the genome's coding and non-coding regions. Many SNPs in some genes (e.g., HSP70, HSP90, HSF1, EIF2AK4, HSBP1, HSPB8, HSPB7, MYO1A, and ATP1A1) in various breeds of cattle have been analyzed to play key roles in many cellular activities during thermal stress and protecting cells against stress, making them potential candidate genes for molecular markers of thermotolerance. This review highlighted the associations of SNPs within these genes with thermotolerance traits (e.g., blood biochemistry and physiological responses), suggesting their potential use as MAS in thermotolerant cattle breeding.
... The acquired thermotolerance of Saccharomyces cerevisiae is a classic example: This yeast is unable to survive at 55 °C if it was cultured at its optimal temperature (28-30 °C) previously. However, it can efficiently adapt to this heat stress after pretreatment at 37 °C (mild heat stress) (Lindquist 1986). Acquired thermotolerance has been observed in several fungi, such as Neurospora crassa, Schizosaccharomyces pombe and C. albicans (Plesofsky-Vig and Tbrambl 1985;De Virgilio et al. 1990;Argüelles 1997), but this phenomenon is not restricted to heat stress. ...
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In the ever-changing fungal environment, fungi have to cope with a wide array of very different stresses. These stresses frequently act in combination rather than independently, i.e., they quickly follow one another or occur concomitantly. Combinatorial stress response studies revealed that the response of fungi to a stressor is highly dependent on the simultaneous action of other stressors or even on earlier stresses to which the fungi adapted. Several important phenomena were discovered, such as stress pathway interference, acquired stress tolerance, stress response memory or stress cross-protection/sensitization, which cannot be interpreted when we study the consequences of a single stressor alone. Due to the interactions between stressors and stress responses, a stress response that develops under a combined stress is not the simple summation of stress responses observed during single stress treatments. Based on the knowledge collected from single stress treatment experiments, we cannot predict how fungi will respond to a certain combination of stresses or even whether this combination will be more harmful than single stress treatments. This uncertainty warns us that if we want to understand how fungi adapt to a certain habitat (e.g., to the human body) to fnd a point of weakness in this adaptation, we must understand how the fungi cope with combinations of stresses, rather than with single stressors.
... Specifically, upper tolerance was greater under fluctuating than constant temperatures (Figures 2 and 3), as it has been reported in other tadpoles and other ectotherms (Barria & Bacigalupe, 2017;Bartheld et al., 2017;Kern et al., 2015;Ramirez et al., 2020;Salachan & Sørensen, 2017;Schou et al., 2017;Simon et al., 2015;Sørensen et al., 2016). Increases in CT max under a regimen of temperature fluctuations could be due to multiple heat shock responses induced by the repeated stressful peak upper temperatures (Lindquist, 1986;Salachan & Sørensen, 2017) or because recovery periods between heat peaks may allow for repair of heat damage (Boardman et al., 2013). Additionally, physiological compensatory changes at the level of enzymes (isoenzymes, chaperones) may probably allow individuals to remain metabolically active over a broader temperature range than those acclimated to constant temperatures (Feldmeth et al., 1974). ...
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Current climate change is generating accelerated increase in extreme heat events and organismal plastic adjustments in upper thermal tolerances, (critical thermal maximum -CTmax ) are recognized as the quicker mitigating mechanisms. However, current research casts doubt on the actual mitigating role of thermal acclimation to face heat impacts, due to its low magnitude and weak environmental signal. Here, we examined these drawbacks by first estimating maximum extent of thermal acclimation by examining known sources of variation affecting CTmax expression, such as daily thermal fluctuation and heating rates. Second, we examined whether the magnitude and pattern of CTmax plasticity is dependent of the thermal environment by comparing the acclimation responses of six species of tropical amphibian tadpoles inhabiting thermally contrasting open and shade habitats and, finally, estimating their warming tolerances (WT = CTmax - maximum temperatures) as estimator of heating risk. We found that plastic CTmax responses are improved in tadpoles exposed to fluctuating daily regimens. Slow heating rates implying longer duration assays determined a contrasting pattern in CTmax plastic expression, depending on species environment. Shade habitat species suffer a decline in CTmax whereas open habitat tadpoles greatly increase it, suggesting an adaptive differential ability of hot exposed species to quick hardening adjustments. Open habitat tadpoles although overall acclimate more than shade habitat species, cannot capitalize this beneficial increase in CTmax, because the maximum ambient temperatures are very close to their critical limits, and this increase may not be large enough to reduce acute heat stress under the ongoing global warming.
... Hsps commonly exist in both prokaryotes and eukaryotes. Based on molecular weight (kDa), Hsps are divided into four types, Hsp90, Hsp70, Hsp60, and small Hsps, which are involved in the transport, folding, unfolding, assembly, and disassembly of multi-structured units, and in the degradation of misfolded or aggregated proteins (Lindquist, 1985;Feder and Hofmann, 1999;Pockley et al., 2007). ...
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Heat shock proteins (Hsps) function as molecular chaperones that enable organisms to withstand stress and maintain normal life activities. In this study, we identified heat shock protein 70 (encoded by hsp70 ), which exhibits a higher expression in the mature male testis than in the unmature testis of Ophraella communa . Tissue expression profile revealed that Ochsp70 levels in males were highest in the testis, whereas those in females were highest in the head. Moreover, the expression of Ochsp70 was found to be significantly induced in female bursa copulatrix after mating. Double-stranded RNA ds Ochsp70 was injected into males to performance RNA interference, which significantly decreased the male Ochsp70 expression levels within 20 d post-injection, whereas no effect was observed on the Ochsp70 expression level in the females after mating with ds Ochsp70 -injected males. However, significant downregulation of female fertility was marked simultaneously. Furthermore, knockdown of female Ochsp70 expression also led to a significant reduction in fertility. Finally, comparative transcriptomic analysis identified glucose dehydrogenase and insulin-like growth factor binding protein as putative downstream targets of Ochsp70 . Overall, we deduced that Ochsp70 is an indispensable gene and a potential male mating factor in O. communa , which regulates reproduction.
... The term "heat shock" proteins was coined as a legacy of Ritossa's pioneering discovery that heat shock produced chromosomal puffs in the salivary glands of Drosophila larvae [4,5]. Later, it was established that the heat-shock response (HSR) is a universal response to an extensive array of stresses [6,7]. HSPs are not only essential during stress, but they are equally crucial in normal conditions to maintain proteostasis [8,9]. ...
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The heat shock protein 90 (Hsp90) is a molecular chaperone and a key regulator of proteostasis under both physiological and stress conditions. In mammals, there are two cytosolic Hsp90 isoforms: Hsp90α and Hsp90β. These two isoforms are 85% identical and encoded by two different genes. Hsp90β is constitutively expressed and essential for early mouse development, while Hsp90α is stress-inducible and not necessary for survivability. These two isoforms are known to have largely overlapping functions and to interact with a large fraction of the proteome. To what extent there are isoform-specific functions at the protein level has only relatively recently begun to emerge. There are studies indicating that one isoform is more involved in the functionality of a specific tissue or cell type. Moreover, in many diseases, functionally altered cells appear to be more dependent on one particular isoform. This leaves space for designing therapeutic strategies in an isoform-specific way, which may overcome the unfavorable outcome of pan-Hsp90 inhibition encountered in previous clinical trials. For this to succeed, isoform-specific functions must be understood in more detail. In this review, we summarize the available information on isoform-specific functions of mammalian Hsp90 and connect it to possible clinical applications.
... The same analysis The expression of HSPs is primarily activated by the heat shock response-associated transcription factor HSF1 in response to pathologic insults that disrupt cytosolic proteostasis, including modest changes in temperature and oxidative stress (45,46). HSPs function to enhance proteostasis capacity of the cell and prevent the pathologic accumulation of potentially toxic protein aggregates (47). Accordingly, DAVID analysis pointed to stress response and response to unfolded protein in the cluster with the highest enrichment score ( Fig. 4B and fig. ...
Article
Disrupted circadian rhythmicity is a prominent feature of modern society and has been designated as a probable carcinogen by the World Health Organization. However, the biological mechanisms that connect circadian disruption and cancer risk remain largely undefined. We demonstrate that exposure to chronic circadian disruption [chronic jetlag (CJL)] increases tumor burden in a mouse model of KRAS-driven lung cancer. Molecular characterization of tumors and tumor-bearing lung tissues revealed that CJL enhances the expression of heat shock factor 1 (HSF1) target genes. Consistently, exposure to CJL disrupted the highly rhythmic nuclear trafficking of HSF1 in the lung, resulting in an enhanced accumulation of HSF1 in the nucleus. HSF1 has been shown to promote tumorigenesis in other systems, and we find that pharmacological or genetic inhibition of HSF1 reduces the growth of KRAS-mutant human lung cancer cells. These findings implicate HSF1 as a molecular link between circadian disruption and enhanced tumorigenesis.
... Later on, HSP70 became inducible which made organisms more thermo tolerant (Browder et al., 1998). However, induction of HSP70 can be inhibited by some inhibitors like cycloheximide which can interfere with normal cellular stress response (Lindquist, 1986). Valenzuela (2000) studied HSP70 expression in five different breeds of cattle and only HSP70 was expressed in lymphocytes. ...
Article
Thermal stress is a unique and complex process which alters the normal physiological mechanisms. Heat shock proteins (HSPs) are known to be highly conserved and ubiquitous proteins synthesized in response to several stimuli. The HSP inducers have been categorized as environmental (heat shock, UV radiation, heavy metals, amino acids, oxidative stress, etc.) pathological (bacterial or parasitic infections, fever, inflammation, etc.) and physiological (growth factors, cell differentiation, hormonal stimulation or tissue development, caloric restriction, etc.). The HSPs play an important role for maintaining cellular functioning under environmental challenges and protein denaturation conditions. There is a great potential for using HSP72 expression to detect natural adaptation and exposure to stress in natural populations. Leptin is a 16 kDa polypeptide hormone secreted primarily from the cells of white adipose tissue. Leptin acts through both central and peripheral mechanisms to affect feeding behaviour, lipid and glucose metabolism, thermogenesis, reproductive and endocrine functions. Leptin also plays an important role in the regulation of body weight and composition, e.g. reduction in feed intake and increase in energy expenditure. Bovine lymphocytes express Ob-Rb gene (leptin receptor) and also express leptin. There are ample information on the central role of leptin in brain and neural tissue regulation. In dairy cattle, the increase in milk yield has been associated with a negative energy balance and a decrease in fertility during early lactation. The hormone leptin is involved in regulation of nutritional status and reproductive function.
... After the discovery of heat shock proteins, it was clear that the heat shock protein response requires a specific transcription factor [40][41][42][43][44]. In crustaceans, a few reports have suggested that the heat shock transcription factor plays a significant role in determining the heat shock response and also in tolerance against stressful conditions. ...
Article
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Heat shock proteins (Hsps) are a family of ubiquitously expressed stress proteins and extrinsic chaperones that are required for viability and cell growth in all living organisms. These proteins are highly conserved and produced in all cellular organisms when exposed to stress. Hsps play a significant role in protein synthesis and homeostasis, as well as in the maintenance of overall health in crustaceans against various internal and external environmental stresses. Recent reports have suggested that enhancing in vivo Hsp levels via non-lethal heat shock, exogenous Hsps, or plant-based compounds, could be a promising strategy used to develop protective immunity in crustaceans against both abiotic and biotic stresses. Hence, Hsps as the agent of being an immune booster and increasing disease resistance will present a significant advancement in reducing stressful conditions in the aquaculture system.
... HSPs have various functions and can help repair and refold denatured proteins back to their native structure after exposure to stressors . The inducible form of HSP70 is one of the most highly conserved chaperones and is thought to have important functions, especially in the tolerance against high temperatures (Feder and Hofmann, 1999;Lindquist, 1986;Nagao et al., 1990). The transcription of HSPs is triggered by the occurrence of denatured proteins in cells (Ananthan et al., 1986) and has been well studied in various organisms (Feder and Hofmann, 1999;Waagner et al., 2010). ...
Article
Climate change has intensified the occurrence of heat waves, resulting in organisms being exposed to thermal and chemical stress at the same time. The effects of mild heat shock combined with sublethal concentrations of phenanthrene (PHE) on defense mechanisms in springtails Folsomia candida were investigated. The transcription of Heat Shock Protein 70 (HSP70) was significantly upregulated by heat shock but tended to reach the control levels after 42 h of recovery. The transcription of cytochrome P450 3A13 (CYP3A13) was upregulated 3-13 fold by PHE but suppressed by heat shock. The suppression by heat shock might contribute to the reduced detoxification of PHE during high-temperature exposure. In line with this, we found that the internal PHE concentration was approximately 70% higher in heat-shocked springtails than in animals kept at control temperature. In general, the transcription of genes encoding enzymes of detoxification phase II (glutathione S-transferase 3) and phase III (ABC transporter 1) and the activity of antioxidant defense enzymes (superoxide dismutase and catalase) were less influenced than genes encoding phase I detoxification mechanisms (CYP3A13). These results indicate that heat shock delays the detoxification of PHE in springtails.
... Ritossa believed that the expansion of chromosomes was the result of the activation of corresponding genes in response to heat stress leading to the increased expression of specific proteins, which are now known to encode proteins called heat shock proteins (HSPs) [2]. HSPs is a group of highly conserved proteins; both the simplest prokaryotes and complex mammals contain HSPs [3]. Unlike other mostly cellular proteins, HSP expression and synthesis are increased following stresses such as oxidative stress, nutrient deficiency, UV radiation, chemicals, viruses, and ischemia-reperfusion injury [4][5][6][7]. ...
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Cardiovascular disease is the result of complicated pathophysiological processes in the tissues that make up the blood vessels and heart. Heat shock protein 90 (HSP90) can interact with 10% of the proteome and is the most widely studied molecular chaperone in recent years. HSP90 is extensively involved in the regulation of protein folding and intracellular protein stability, making HSP90 a hopeful target for the treatment of multiple cardiovascular diseases. Numerous client proteins of HSP90 have been identified in known cardiac disease pathways, including MAPK signaling, PI3K/AKT (PKB)/mTOR, and TNF-α signaling. Therefore, these pathways can be controlled by regulating HSP90. Among them, the activity of HSP90 can be regulated via numerous inhibitors. In this review, first, we will discuss the function of HSP90 and its role in pathological pathways. In addition, HSP90 plays a significant role in most cardiovascular diseases, including hypertension, pulmonary venous hypertension, atherosclerosis, and heart failure; next we will focus on this part. Finally, we will summarize the currently known HSP90 inhibitors and their potential in the treatment of heart disease.
... Based on our findings, we hypothesize that the absence of Hal9 preactivates the Msn2/4 response and upregulates Hsp12 levels giving the cells the ability to survive the subsequent challenge by MMV688766. This is similar to how in the well-conserved phenomenon of thermotolerance, prior nontoxic heat stress enables cells to tolerate subsequent, otherwise lethal heat stress (70,71). A recent large-scale investigation of adaptive evolution to xenobiotics revealed that SNPs in HAL9 confer resistance to other molecules, specifically loratadine and MMV085203, in the same S. cerevisiae drug-sensitized background that was used in this work (46). ...
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Candida species are among the most prevalent causes of systemic fungal infection, posing a growing threat to public health. While Candida albicans is the most common etiological agent of systemic candidiasis, the frequency of infections caused by non-albicans Candida species is rising. Among these is Candida auris, which has emerged as a particular concern. Since its initial discovery in 2009, it has been identified worldwide and exhibits resistance to all three principal antifungal classes. Here, we endeavored to identify compounds with novel bioactivity against C. auris from the Medicines for Malaria Venture's Pathogen Box library. Of the five hits identified, the trisubstituted isoxazole MMV688766 emerged as the only compound displaying potent fungicidal activity against C. auris, as well as other evolutionarily divergent fungal pathogens. Chemogenomic profiling, as well as subsequent metabolomic and phenotypic analyses, revealed that MMV688766 disrupts cellular lipid homeostasis, driving a decrease in levels of early sphingolipid intermediates and fatty acids and a concomitant increase in lysophospholipids. Experimental evolution to further probe MMV688766's mode of action in the model fungus Saccharomyces cerevisiae revealed that loss of function of the transcriptional regulator HAL9 confers resistance to MMV688766, in part through the upregulation of the lipid-binding chaperone HSP12, a response that appears to assist in tolerating MMV688766-induced stress. The novel mode of action we have uncovered for MMV688766 against drug-resistant fungal pathogens highlights the broad utility of targeting lipid homeostasis to disrupt fungal growth and how screening structurally-diverse chemical libraries can provide new insights into resistance-conferring stress responses of fungi. IMPORTANCE As widespread antimicrobial resistance threatens to propel the world into a postantibiotic era, there is a pressing need to identify mechanistically distinct antimicrobial agents. This is of particular concern when considering the limited arsenal of drugs available to treat fungal infections, coupled with the emergence of highly drug-resistant fungal pathogens, including Candida auris. In this work, we demonstrate that existing libraries of drug-like chemical matter can be rich resources for antifungal molecular scaffolds. We discovered that the small molecule MMV688766, from the Pathogen Box library, displays previously undescribed broad-spectrum fungicidal activity through perturbation of lipid homeostasis. Characterization of the mode of action of MMV688766 provided new insight into the protective mechanisms fungi use to cope with the disruption of lipid homeostasis. Our findings highlight that elucidating the genetic circuitry required to survive in the presence of cellular stress offers powerful insights into the biological pathways that govern this important phenotype.
... The clinical potential of hyperthermia is attenuated by the ability of (cancer) cells to activate a protective mechanism known as the heat stress response (HSR) [10,11]. First, activation of the HSR confers thermotolerance-transient resistance to successive heat exposures-which has been found to reduce thermo-sensitization [12,13], and is among the major reasons for generally limiting the frequency of therapeutic hyperthermia sessions to one or two per week [14]. ...
Article
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Hyperthermia is being used as a radio- and chemotherapy sensitizer for a growing range of tumor subtypes in the clinic. Its potential is limited, however, by the ability of cancer cells to activate a protective mechanism known as the heat stress response (HSR). The HSR is marked by the rapid overexpression of molecular chaperones, and recent advances in drug development make their inhibition an attractive option to improve the efficacy of hyperthermia-based therapies. Our previous in vitro work showed that a single, short co-treatment with a HSR (HSP90) inhibitor ganetespib prolongs and potentiates the effects of hyperthermia on DNA repair, enhances hyperthermic sensitization to radio- and chemotherapeutic agents, and reduces thermotolerance. In the current study, we first validated these results using an extended panel of cell lines and more robust methodology. Next, we examined the effects of hyperthermia and ganetespib on global proteome changes. Finally, we evaluated the potential of ganetespib to boost the efficacy of thermo-chemotherapy and thermo-radiotherapy in a xenograft murine model of cervix cancer. Our results revealed new insights into the effects of HSR inhibition on cellular responses to heat and show that ganetespib could be employed to increase the efficacy of hyperthermia when combined with radiation.
... For instance, the cellular stress response to high temperature encompasses an overall decline in transcription and translation and enhanced degradation of damaged or misfolded proteins. Concurrently, the expression of stress response genes, such as heat shock protein (HSP) coding genes, is elevated (1)(2)(3). Usually, after the culmination of stress, cells recuperate by restoring the gene expression status to that of a standard scenario. However, emerging evidence suggests that specific stress-induced changes are not only remembered throughout the lifetime of an organism but are also inherited by subsequent generations, having the potential to be adaptive or maladaptive. ...
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The notion that genes are the sole units of heredity and that a barrier exists between soma and germline has been a major hurdle in elucidating the heritability of traits that were observed to follow a non-Mendelian inheritance pattern. It was only after the conception of epigenetics by Conrad Waddington that the effect of parental environment on subsequent generations via non-DNA sequence-based mechanisms, such as DNA methylation, chromatin modifications, non-coding RNAs, and proteins, could be established, now referred to as multigenerational epigenetic inheritance. Despite growing evidence, the male gamete-derived epigenetic factors that mediate the transmission of such phenotypes are seldom explored, particularly in the model organism Drosophila melanogaster. Using the heat stress-induced multigenerational epigenetic inheritance paradigm in a widely used position-effect variegation line of Drosophila, named white-mottled, we have dissected the effect of heat stress on the sperm proteome in the current study. We demonstrate that multiple successive generations of heat stress at the early embryonic stage results in a significant downregulation of proteins associated with a diverse set of functions, such as translation, chromatin organization, microtubule-based processes, and generation of metabolites and energy, in the sperms. Based on our findings, we propose chromatin-based epigenetic mechanisms, a well-established mechanism for multigenerational effects, as a plausible way of transmitting heat stress memory via the male germline in this case. Moreover, we show that despite these heat stress-induced changes, the life-history traits, such as reproductive fitness and stress tolerance of the subsequent generations, are unaffected, probing the evolutionary relevance of multigenerational epigenetic effects.
... Heat shock proteins(HSPs) are a family of proteins that were discovered in Drosophila after exposure to high temperatures caused the genes to appear as chromosome puffs, hence the name heat shock in 1962 (Ritossa, 1962). Subsequently, many HSPs and their genes have been characterized (Fehrenbach and Niess, 1999;Lindquist 2003;Sørensen et al., 2003). HSPs are highly conserved proteins which are found in all organisms from bacteria to plants and animals (Kiang and Tsokos, 1998;Sørensen et al., 2003). ...
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Mizuhopecten yessoensis, one of the most highly favored scallops on the international market, has suffered from massive summer mortalities. Water temperature is an important environmental stressor which has significant effects on the physiological and biochemical response of scallops. Heat shock protein 90 (HSP90) plays a key role in defense against various environmental stresses that could damage the cellular and molecular structure of cells. In this study, molecular characterization and expression of HSP90 in M. yessoensis (designated MyHSP90) were analyzed as an indicator to understand the mechanisms of heat shock response in M. yessoensis under temperature stress. The full-length sequence of MyHSP90 cDNA (GenBank accession no. MF196912) is composed of 2639 base pair encoding a 726-amino acid polypeptide with a predicted molecular mass of 83.26 kDa. Tissue expression analysis of MyHSP90 genes revealed ubiquitous expression in each tissue examined, and showed a temperature-dependent response, and the expression level was up-regulated significantly in all the six tissues tested (p < 0.05) except gonad. The results will be useful in furthering the understanding of the massive summer mortalities in M. yessoensis under temperature stress. Additionally, MyHSP90 could be used as a potential biomarker in practice to monitor environmental changes in future studies.
... The transcriptional response to proteotoxic stress was uncovered in a serendipitous observation by Ritossa in the 1960s [8]. Heat-shock puffs observed on drosophila (Drosophila melanogaster) salivary gland polytene chromosomes represented the induction of chaperones such as HSP70 and HSP90 [9,10]. The rapid upregulation of chaperone genes has been extensively studied in the past two decades, providing insightful mechanisms of stress sensing and transcriptional response [11]. ...
Article
Cells experiencing proteotoxic stress downregulate the expression of thousands of active genes and upregulate a few stress-response genes. The strategy of downregulating gene expression has conceptual parallels with general lockdown in the global response to the coronavirus disease 2019 (COVID-19) pandemic. The mechanistic details of global transcriptional downregulation of genes, termed stress-induced transcriptional attenuation (SITA), are only beginning to emerge. The reduction in RNA and protein production during stress may spare proteostasis capacity, allowing cells to divert resources to control stress-induced damage. Given the relevance of translational downregulation in a broad variety of diseases, the role of SITA in diseases caused by proteotoxicity should be investigated in future, paving the way for potential novel therapeutics.
... Heat shock proteins (Hsps) are a group of highly conserved proteins that mitigate the effects of fluctuating environmental conditions by maintaining intracellular protein homeostasis [1][2][3]. Hsps are found in almost all types of cells and tissues in multicellular organisms [4]. Constitutively expressed Hsps are involved in protein folding, intracellular transport, regulation of signal transduction pathways, and cytoskeleton assembly, all of which are essential for normal growth and development [5]. ...
Article
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Anoplophora glabripennis (Agla) is an important global quarantine pest due to its highly destructive impacts on forests. It is widely distributed in many countries in Asia, Europe, and North America. The survival of A. glabripennis larvae has been facilitated by its high adaptability to low temperature. When insects are subjected to temperature stress, heat shock proteins (Hsps) limit cell damage and improve cell tolerance via their protein folding, localization, and degradation activities. However, the temperature adaptation mechanisms of A. glabripennis Hsps remain unclear. In this study, four A. glabripennis Hsp genes, AglaHsp20.43, AglaHsp71.18, AglaHsp82.09, and AglaHsp89.76, were cloned. Sequence analysis showed that all four Hsps had specific conserved domains. Phylogenetic analysis revealed that Hsps from different subfamilies were evolutionarily conserved, and that AglaHsps were highly similar to those of Coleoptera species. Protein expression vectors (pET30a-AglaHsps) were constructed and used to express AglaHsps in E. coli, where all four proteins were expressed in inclusion bodies. Western blot analysis showed that AglaHsps were expressed at a range of temperatures, from −10 °C to 25 °C. AglaHsp82.09 and AglaHsp89.76 showed high expressions with treatment at 0 °C. Our results will facilitate clarification of the molecular mechanisms underlying A. glabripennis responses to environmental stress.
... Altogether, these harmful effects lead to an arrest of the cell cycle and decreased cellular growth and proliferation (Lindquist, 1986). If the HS duration is prolonged or the increase of the temperature is drastic, the accumulation of cellular damages can result in cell death and can further alter the organismal microenvironment. ...
Thesis
Over the years numerous studies unraveled the fascinating ability of the heat stress response (HSR) to act on the genome and to transiently control the cell proteome in order to preserve cell viability in the context of hostile environments. Nevertheless, the implementation and orchestration of the HSR today still hide mysteries. The implication of N6-methyladenosine (m6A) -the most abundant internal RNA modification- in the regulation of gene expression in response to stress has been recently uncovered. Yet, its importance, the associated functions and the involved mechanisms remain poorly characterized.During my PhD thesis I have studied the role of the protein YTHDC1, the main known nuclear reader of m6A, in the conserved cellular stress response to heat shock (HS). Therefore, my thesis work is at the crossroad of two main research axes- the emerging field of the epitranscriptome and the broadly investigated domain of stress biology. In this work, we were able to identify the central role played by YTHDC1, and by extension of the m6A signaling, in the regulation of the HSR pathway in human cells. First, we have demonstrated that during the recovery period following HS YTHDC1 massively relocalizes to characteristic nuclear structures appearing upon stress, named nuclear Stress Bodies (nSBs). This striking relocalization relies on the transcription of repetitive pericentric heterochromatin regions that produces the lncRNAs SATIII, which are at the core of nSBs formation. Importantly, we found out that beyond localizing at nSBs, YTHDC1, together with SATIII lncRNAs, contributes to the stress-induced regulation of alternative splicing events of potentially several hundreds of mRNAs. Second, by conducting ChIP-seq experiments we discovered that, upon HS, YTHDC1 is recruited to new genomic sites. Noticeably, we identified many Heat Shock Protein (HSP)-coding genes to be targets of YTHDC1 upon stress. Follow-up analyses revealed that YTHDC1 is essential for the induction of HSPs expression following HS. The investigation of YTHDC1 molecular functions showed that, under heat stress, the protein may regulate proper transcription termination of HSP genes and the following nuclear export of their mRNAs. Moreover, we found that, in response to stress, YTHDC1 is required to maintain the structure of Nuclear Speckles, which are membrane-less bodies contributing to the control of mRNAs fate. Finally, m6A-dependency of YTHDC1 molecular and cellular functions was addressed in this work through the establishment of a genetically manipulated cellular model. This approach demonstrated that some of the roles of YTHDC1 that we identified are associated with its ability to recognize the m6A RNA mark.In conclusion, this work has uncovered the epitranscriptome reader YTHDC1 as a novel and central regulator of the HSR acting at various levels of the stress-induced genome reprogramming in human cells. Our findings expand the current knowledge on the implementation of stress responses with the addition of a broadly acting layer of gene expression control and open new area of research for future studies.
... HSPs are involved in responses to diverse sources of stress that disrupt protein conformation, such as ethanol, arsenite, cadmium, zinc, copper, mercury, sulfhydryl reagents, calcium ionophores, steroid hormones, chelating agents, and viruses [68]. ...
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Coronaviruses have been responsible for multiple challenging global pandemics, including coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Papain-like protease (PLpro), one of two cysteine proteases responsible for the maturation and infectivity of SARS-CoV-2, processes and liberates functional proteins from the viral polyproteins and cleaves ubiquitin and ISG15 modifications to inhibit innate immune sensing. Consequently, PLpro is an attractive target for developing COVID-19 therapies. PLpro contains a zinc-finger domain important for substrate binding and structural stability. However, the impact of metal ions on the activity and biophysical properties of SARS-CoV-2 PLpro has not been comprehensively studied. Here, we assessed the impacts of metal ions on the catalytic activity of PLpro. Zinc had the largest inhibitory effect on PLpro, followed by manganese. Calcium, magnesium, and iron had smaller or no effects on PLpro activity. EDTA at a concentration of 0.5 mM was essential for PLpro activity, likely by chelating trace metals that inhibit PLpro. IC50 values for ZnCl2, ZnSO4, and MnCl2 of 0.42 ± 0.02 mM, 0.35 ± 0.01 mM, and 2.6 ± 0.3 mM were obtained in the presence of 0.5 mM EDTA; in the absence of EDTA, the estimated IC50 of ZnCl2 was 14 µM. Tryptophan intrinsic fluorescence analysis confirmed the binding of zinc and manganese to PLpro, and differential scanning calorimetry revealed that zinc but not manganese reduced ΔHcal of PLpro. The results of this study provide a reference for further work targeting PLpro to prevent and treat COVID-19.
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Rainbow trout ( Oncorhynchus mykiss ) is a cold-water fish that is commonly harmed by high temperatures. MicroRNAs (miRNAs) are being investigated intensively because they act as essential metabolic regulators and have a role in the heat stress response. Although there have been numerous studies on rainbow trout heat stress, research on miRNA implicated in rainbow trout heat stress is quite restricted. Rainbow trout were sampled at 18 and 24 °C, respectively, to examine the mechanism of miRNA under heat stress, and we identified a heat stress-induced miRNA, ssa-miR-301a-3p, for further investigation based on our bioinformatics analysis of rainbow trout small RNA sequencing data. Bioinformatics research suggested that hsp90b2 is a probable target gene for ssa-miR-301a-3p. QRT-PCR was used to confirm the expression levels of ssa-miR-301a-3p and hsp90b2 . Meanwhile, the dual-luciferase reporter assay was employed to validate the ssa-miR-301a-3p -hsp90b2 targeted connection. The results indicated that at 24 °C, the relative expression of ssa-miR-301a-3p was considerably lower than at 18 °C. On the other hand, hsp90b2 expression, followed the opposite pattern. The binding of ssa-miR-301a-3p to the 3′-UTR of hsp90b2 resulted in a substantial decrease in luciferase activity. The findings showed that ssa-miR-301a-3p was implicated in heat stress, and our findings provide fresh insights into the processes of miRNA in response to heat stress in rainbow trout.
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Background Thermoinducible bioswitches are unique in that the all-or-none switch response is triggered by temperature, which is a global factor that impacts all biochemical reaction processes. To date, temperature-inducible bioswitches rely exclusively on special thermal sensing biomolecules of DNA, RNA, proteins and lipids whose conformations are critically temperature dependent. Method This paper extends the traditional thermal switch by utilizing purposely designed network topologies of biomolecular interactions to achieve the switching function. By assuming the general Arrhenius law for biochemical reactions, we explore the full space of all three-node genetic interaction networks to screen topologies capable of thermal bioswitches. Three target bioswitches, i.e., thermal-inducible Off–On, cold-inducible On–Off, and hybrid Off–On-Off double switches, are considered separately. Conclusions We identify the minimal and core network skeletons that are basic and essential for building robust high-performance bioswitches: three Off–On motifs, three On–Off motifs, and an incoherent feedforward motif for an Off–On-Off double switch. Functional topologies are implicitly preferential in choosing parameter values to achieve the target functions. The scenario of the topology-based bioswitch we propose here is an extension of molecule-based bioswitches and would be valuable in aiding the rational design and synthesis of efficient high-performance thermal bioswitches.
Article
Hippocampus erectus inhabiting the shallow coastal waters of the southern Gulf of Mexico are naturally exposed to marked temperature variations occurring in different temporal scales. Under such heterogeneous conditions, a series of physiological and biochemical adjustments take place to restore and maintain homeostasis. This study investigated the molecular mechanisms involved in the response of H. erectus to increased temperature using transcriptome analysis based on RNA-Seq technology. Data was obtained from seahorses after 0.5-h exposure to combinations of different target temperatures (26 °C: control, and increased to 30 and 33 °C) and rates of thermal increase (abrupt: < 5 min; gradual: 1–1.5 °C every 3 h). The transcriptome of seahorses was assembled de novo using Trinity software to obtain 29,211 genes and 30,479 transcripts comprising 27,520,965 assembled bases. Seahorse exposure to both 30 and 33 °C triggered characteristic processes of the cellular stress response, regardless of the rate of thermal change. The transcriptomic profiles of H. erectus suggest an arrest of muscle development processes, the activation of heat shock proteins, and a switch to anaerobic metabolism within the first 0.5 h of exposure to target temperatures to ensure energy supply. Interestingly, apoptotic processes involving caspase were activated principally in gradual treatments, suggesting that prolonged exposure to even sublethal temperatures results in the accumulation of deleterious effects that may eventually terminate in cellular death. Results herein validate 30 °C and 33 °C as potential upper limits of thermal tolerance for H. erectus at the southernmost boundary of its geographic distribution.
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Background Glioblastoma is the most aggressive form of brain cancer, characterised by high proliferation rates and cell invasiveness. Despite advances in surgery and radio-chemotherapy, patients continue to have poor prognoses, with a survival rate of 14–15 months. Thus, new therapeutic strategies are needed. Non-ionising electromagnetic fields represent an emerging option given the potential advantages of safety, low toxicity and the possibility to be combined with other therapies. Methods Here, the anticancer activity of quantum molecular resonance (QMR) was investigated. For this purpose, three glioblastoma cell lines were tested, and the QMR effect was evaluated on cancer cell proliferation rate and aggressiveness. To clarify the QMR mechanism of action, the proteomic asset after stimulation was delineated. Mesenchymal stromal cells and astrocytes were used as healthy controls. Results QMR affected cancer cell proliferation, inducing a significant arrest of cell cycle progression and reducing cancer tumorigenicity. These parameters were not altered in healthy control cells. Proteomic analysis suggested that QMR acts not only on DNA replication but also on the machinery involved in the mitotic spindle assembly and chromosome segregation. Moreover, in a combined therapy assessment, QMR significantly enhanced temozolomide efficacy. Conclusions QMR technology appears to be a promising tool for glioblastoma treatment.
Article
Soybean cyst nematode (SCN) Heterodera glycines is considered as the major constraint to soybean production. GmSHMT08 at Rhg4 locus on chromosome 08, encoding a serine hydroxylmethyltransferase, is a major gene underlying resistance against H. glycines in Peking-type soybeans. However, the molecular mechanism underpinning this resistance is less well characterized, and whether GmSHMT08 could interact with proteins in H. glycines remains unclear. In this study, yeast two-hybrid screening was conducted using GmSHMT08 as a bait protein, and a fragment of a 70kDa heat shock protein (HgHSP70) was screened from H. glycines that exhibited interaction with GmSHMT08. This interaction was verified by both GST pull-down and bimolecular fluorescence complementation assays. Our finding reveals HgHSP70 could be applied as an essential candidate gene for further exploring the mechanism on GmSHMT08-mediated resistance against SCN H. glycines.
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Heat shock promotes the assembly of translation factors into condensates to facilitate the production of stress-protective proteins. How translation factors detect heat and assemble into condensates is not well understood. Here, we investigate heat-induced condensate assembly by the translation factor Ded1p from five different fungi, including Ded1p from Saccharomyces cerevisiae. Using targeted mutagenesis and in vitro reconstitution biochemistry, we find that heat-induced Ded1p assembly is driven by a conformational rearrangement of the folded helicase domain. This rearrangement determines the assembly temperature and the assembly of Ded1p into nanometer-sized particles, while the flanking intrinsically disordered regions engage in intermolecular interactions to promote assembly into micron-sized condensates. Using protein engineering, we identify six amino acid substitutions that determine most of the thermostability of a thermophilic Ded1p ortholog, thereby providing a molecular understanding underlying the adaptation of the Ded1p assembly temperature to the specific growth temperature of the species. We conclude that heat-induced assembly of Ded1p into translation factor condensates is regulated by a complex interplay of the structured domain and intrinsically disordered regions which is subject to evolutionary tuning.
Chapter
Purpose of review: Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR) in mammalian cells and is a critical element in maintaining protein homeostasis. HSF1 functions at the center of many physiological processes like embryogenesis, metabolism, immune response, aging, cancer, and neurodegeneration. However, the mechanisms that allow HSF1 to control these different biological and pathophysiological processes are not fully understood. This review focuses on Huntington's disease (HD), a neurodegenerative disease characterized by severe protein aggregation of the huntingtin (HTT) protein. The aggregation of HTT, in turn, leads to a halt in the function of HSF1. Understanding the pathways that regulate HSF1 in different contexts like HD may hold the key to understanding the pathomechanisms underlying other proteinopathies. We provide the most current information on HSF1 structure, function, and regulation, emphasizing HD, and discussing its potential as a biological target for therapy. Data sources: We performed PubMed search to find established and recent reports in HSF1, heat shock proteins (Hsp), HD, Hsp inhibitors, HSF1 activators, and HSF1 in aging, inflammation, cancer, brain development, mitochondria, synaptic plasticity, polyglutamine (polyQ) diseases, and HD. Study selections: Research and review articles that described the mechanisms of action of HSF1 were selected based on terms used in PubMed search. Results: HSF1 plays a crucial role in the progression of HD and other protein-misfolding related neurodegenerative diseases. Different animal models of HD, as well as postmortem brains of patients with HD, reveal a connection between the levels of HSF1 and HSF1 dysfunction to mutant HTT (mHTT)-induced toxicity and protein aggregation, dysregulation of the ubiquitin-proteasome system (UPS), oxidative stress, mitochondrial dysfunction, and disruption of the structural and functional integrity of synaptic connections, which eventually leads to neuronal loss. These features are shared with other neurodegenerative diseases (NDs). Currently, several inhibitors against negative regulators of HSF1, as well as HSF1 activators, are developed and hold promise to prevent neurodegeneration in HD and other NDs. Conclusion: Understanding the role of HSF1 during protein aggregation and neurodegeneration in HD may help to develop therapeutic strategies that could be effective across different NDs.
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Food security is a global challenge and present staple foods are not sufficient to fulfill the increasing demand for food for growing population in terms of yield as well as nutrition. Small millets are nutritionally rich and have the potential to become the new staple. Small millets are a group of crops comprising finger millet, kodo millet, little millet, foxtail millet, barnyard millet, and proso millet. The crops are grown in a variety of agroecological conditions in diverse soils and varying rainfall. The growth and nutrition of the crops are highly influenced by abiotic stresses. Thus, majority of the crop improvement programs focus on the identification and development of stress tolerance mechanisms in crops. However, most of the crop improvement stratagems are carried out in staple cereals such as rice, wheat, and maize. Whereas very less or negligible attention is given toward minor millets. The present chapter summarizes the efforts carried out to explore the insights into abiotic stress tolerance on small millets through a transcriptomics approach.
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With climate change a reality today, towards maintaining a sustainable and enhanced production we require crop plants well suited to stressful environments. Small millets, hitherto relegated away from the mainstream agriculture are slowly and steadily making a comeback in recent times. This diverse group of crop plants are a veritable powerhouse of genomic resource for mining stress tolerant genes and alleles with their natural inherent resilience. One such group of molecules are the multifaceted molecular chaperones called heat shock proteins (HSPs). From their initial discovery in Drosophila in 1962, HSPs have come a long way as a multi-functional group of proteins with demonstrated critical roles in plant life cycle, hormonal biology as well as regulatory role in plant abiotic and biotic defence. In this chapter we have described the structural and functional attributes of HSP in model plants and small millets, thereby highlighting their potential roles. The research can be utilized towards improvement of small millets as well as other crop plants for multiple stress tolerance.
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The mulberry silkworm, Bombyx mori L., is an economically important insect reared indoors for producing silk in several countries. Advances made in several aspects of silkworm genetic improvement are reviewed, including raw silk production, silkworm genomic and genetic resources, silkworm breed improvement programmes, breeding methods and silkworm breeds/hybrids (bivoltine and polyvoltine), which are covered in this chapter. Breeding for resistance/tolerance to abiotic (high temperature, high humidity, fluoride pollution) and biotic (BmNPV, BmBDV, BmIFV) stress and molecular approaches/strategies being utilized for silkworm breed improvement across the world is also included. This chapter provides insights into studies on genetic improvement for commercial exploitation of mulberry silkworm hybrids and future perspectives.KeywordsMulberry silkwormBreeding and geneticsMolecular approachesGenetic manipulation
Article
During the heat shock response (HSR), heat shock factor (HSF1 in mammals) binds to target gene promoters, resulting in increased expression of heat shock proteins that help maintain protein homeostasis and ensure cell survival. Besides HSF1, only a relatively few transcription factors with a specific role in ensuring correctly regulated gene expression during the HSR have been described. Here, we use proteomic and genomic (CRISPR) screening to identify a role for RPRD1B in the response to heat shock. Indeed, cells depleted for RPRD1B are heat shock sensitive and show decreased expression of key heat shock proteins (HSPs). These results add to our understanding of the connection between basic gene expression mechanisms and the HSR.
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Heat shock proteins (HSPs) are a kind of proteins which mostly found in bacterial, plant and animal cells, in which they are involved in the monitoring and regulation of cellular life activities. HSPs protect other proteins under environmental and cellular stress by regulating protein folding and supporting the correctly folded structure of proteins as chaperones. During viral infection, some HSPs can have an antiviral effect by inhibiting viral proliferation through interaction and activating immune pathways to protect the host cell. However, although the biological function of HSPs is to maintain the homeostasis of cells, some HSPs will also be hijacked by viruses to help their invasion, replication, and maturation, thereby increasing the chances of viral survival in unfavorable conditions inside the host cell. In this review, we summarize the roles of the heat shock protein family in various stages of viral infection and the potential uses of these proteins in antiviral therapy.
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Functional characters of polythene chromosomes andchromosomal rearrangements in salivary glands of 177 larvae of Glyptotendipesglaucus (Diptera, Chironomidae) from reservoirs of Russiaand Ukraine (Chernobyl) have been analysed. Similarity of the populationsstudied based on a pool of chromosomal reorganizations has beenestablished. The general types of inversions in chromosomal arms A, B, Dand E have been detected. Influence of radioactive pollution (Chernobyl)on functional changes of a nucleus, Balbiani rings, puffs, morphology ofdisks and interdisks is revealed.
Article
Zinc supplementation prior to heat shock increases HSP70 (heat shock protein 70) expression, which has cytoprotective effects in tissue cells during inflammation. Effects of zinc deficiency in this regard have been discussed controversially. Whether zinc modulates the expression of HSP70 in the human immune system as well and thus affects cell survival during heat stress is so far largely unknown. Therefore, we investigated the effect of alterations in the cellular zinc status on HSP70 expression and on cellular survival in human monocytes and lymphocytes. Three cell lines (Jurkat, THP-1, and Ramos) and enriched primary human monocytes and lymphocytes from young subjects were subjected to zinc deficiency or supplementation and subsequently heat shock at 42 °C. HSP70 mRNA expression was analyzed by real-time PCR, whereas HSP70 protein expression was analyzed by western blotting. In all cells other than Ramos cells, zinc supplementation and deficiency augmented heat shock-induced HSP70 expression. Further experiments in primary monocytes and lymphocytes indicated that this may be explained by the enhanced phosphorylation of HSF1 (Heat shock factor 1) at Ser326, which plays a significant role in HSP70 induction, as observed in zinc deficient and supplemented cells. While zinc supplementation had negligible effects on cell viability, acute zinc deficiency further increased cell death, induced by heat shock. Our results emphasize the importance of an optimal cellular zinc status. Moreover, we present a possible mechanism behind zinc's influence on HSP70 expression in human leukocytes. Our data form the basis for further in vivo and ex vivo studies to investigate how the zinc status may affect cellular damage in transient high temperature situations.
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We have identified and characterized a ribonucleoprotein structure from the cytoplasm of Drosophila melanogaster tissue culture cells which is equivalent to the prosome, a recently described ribonucleoprotein particle of duck and mouse cells. During the recovery period following heat shock, the low mol. wt. heat-shock proteins form cytoplasmic ribonucleoprotein particles which co-purify with the Drosophila prosome. Both ribonucleoprotein particles share several structural properties but their protein constituents differ in their metabolism and cellular localization during the heat treatment. We also report the partial nucleotide sequences of several small RNA species associated with the Drosophila prosome. One of them has a strong sequence homology with the U6 mammalian small nuclear RNA.
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Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.
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Primary isolates of thymic lymphocytes maintained in vitro provide a physiologically well-characterized system in which to study induction of proteins by heat shock; this response is agent-specific and separable from inductions by glucocorticoids or heavy metals (Maytin, E. V., and Young, D. A. J. Biol. Chem. 258, 12718-12722). Here we identify 68 heat shock protein inductions among more than 2,500 individual proteins separated on giant two-dimensional gels and further describe their time course of appearance, sensitivity to cordycepin (3'-deoxyadenosine), and reversibility during recovery. Thirty-one changes are detectable within 1 h. Among these early increases, 20 are inhibitable by cordycepin. However, 11 early changes are not affected by cordycepin; all represent proteins found in relatively low abundance. Five of these inductions are rapidly reversible during recovery from heat shock, in contrast to most other heat shock proteins whose synthesis is maintained or enhanced. One protein identified here appears to be increased by recovery per se. Overall, these results provide evidence for two separate classes of heat shock inductions in normal mammalian cells, i.e. a transcriptionally regulated group, not readily reversible during recovery, and a translationally regulated group in which several inductions rapidly revert to normal during recovery.
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Two phosphoproteins are adsorbed to protein-A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98-100-kDa phosphoprotein that contains the steroid-binding site and the other is a 90-kDa nonsteroid-binding phosphoprotein that is associated with the untransformed, molybdate-stabilized receptor (Housley, P. R., Sanchez, E. R., Westphal, H.M., Beato, M., and Pratt, W.B. (1985) J. Biol. Chem. 260, in press). In this paper we show that the 90-kDa receptor-associated phosphoprotein is an abundant cytosolic protein that reacts with a monoclonal antibody that recognizes the 90-kDa phosphoprotein that binds steroid receptors in the chicken oviduct. The 90-kDa protein immunoadsorbed from L-cell cytosol with this antibody reacts on Western blots with rabbit antiserum prepared against the 89-kDa chicken heat shock protein. Immunoadsorption of molybdate-stabilized cytosol by antibodies against the glucocorticoid receptor results in the presence of a 90-kDa protein that interacts on Western blots with the antiserum against the chicken heat shock protein. The association between the 90-kDa protein and the receptor is only seen by this technique when molybdate is present to stabilize the complex; and when steroid-bound receptors are incubated at 25 degrees C to transform them to the DNA-binding state, the 90-kDa protein dissociates. These observations are consistent with the proposal that the untransformed glucocorticoid receptor in L-cells exists in a complex with the murine 90-kDa heat shock protein.
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A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.
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We have examined in greater detail the derepression of uridine diphosphate galactose-4-epimerase synthesis in capR mutants of Escherichia coli first observed by Markovitz. (Markovitz, A. (1964) Proc. Nat. Acad. Sci. U. S. A. 51, 239–246). All three enzymes of the gal operon are derepressed from 2- to 4-fold by this mutation. This derepression may be superimposed on the conventional derepression seen when the gal operon is induced with fucose or by a regulatory mutation. Measurement of the level of gal messenger ribonucleic acid indicates that the derepression caused by the capR mutation probably occurs at the level of transcription since there is a coordinate increase in gal enzyme activity and in the level of gal mRNA. The effect of the capR mutation on the gal operon appears to be independent of cyclic 3′,5′-monophosphate or glucose-mediated repression. Thus the product of the capR locus behaves as if it is an additional negative control element affecting the expression of the gal operon.
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Using the microcloning approach a number of recombinant lambda phages carrying DNA from the 93D region have been isolated. Screening genomic libraries, cloned in phage lambda or cosmid vectors, with this isolated DNA yielded a series of overlapping DNA fragments from the region 93D6-7 as shown by in situ hybridization to polytene chromosomes. In vitro 32P-labelled nuclear RNA prepared from heat-shocked third instar larvae hybridized specifically to one fragment within 85 kb of cloned DNA. The region which is specifically transcribed after heat shock could be defined to a cluster of internally-repetitive DNA and its neighbouring proximal sequences. Over a sequence of 10-12 kb in length the DNA is cut into repeat units of approximately 280 nucleotides by the restriction endonuclease TaqI. The TaqI repeat sequences are unique in the Drosophila genome.
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Cells having the temperature-sensitive mutation groES131(Ts) were isolated from Escherichia coli K-12 strain C600T by thymineless death selection at 44 degrees C. This conditionally expressed mutation affected both cellular DNA and RNA syntheses at nonpermissive temperature, in addition to rendering cells unable to propagate phage lambda at permissive temperature.
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A novel ribonucleoprotein (RNP) particle showing a highly compact and characteristic structure in the electron microscope was found associated with globin and other repressed mRNA in the cytoplasm of duck, mouse and HeLa cells. This 19S complex is of extraordinary stability: dissociated by 0.5 M KCl or EDTA from the (still repressed) core globin mRNP, it can be purified on gradients containing 1% Sarkosyl, and resists (unfixed) caesium sulphate-dimethylsulphoxide density centrifugation. Its density of 1.31 g/cm3 indicates an RNP complex with a 15% RNA component. In mouse and duck it contains approximately 10 proteins in the 20 000-30 000 mol. wt. range, a few components of 50 000-70 000 mol. wt., and two specific small cytoplasmic RNAs (ScRNA) of 70-90 nucleotides. Both of these RNAs have identical 3'-terminal oligonucleotides. We propose the name 'prosome' for this ScRNP particle which somehow participates in negative control of mRNA translation, and we believe will prove to be ubiquitous to animal species.
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The transforming protein of Rous sarcoma virus (RSV), pp60src, was previously shown to associate with two cellular proteins of Mr 90,000 and 50,000 in RSV-transformed chicken cells. In this report, we demonstrate that this interaction is specific for a discrete population of pp60src molecules. Newly synthesized pp60src was found to preferentially associate with pp90 and pp50 to form a short-lived complex. The half-life of this complex varied from 9 to 15 min in cells transformed by nondefective strains of RSV. This interaction between pp60src, pp50, and pp90 took place in a soluble fraction of the cell, and the complex-bound pp60src molecules were not phosphorylated on tyrosine. These results suggest that pp90 and pp50 may be involved in the processing of pp60src molecules before the association of pp60src with the plasma membrane. The kinetics of dissociation of this complex were shown to be altered in cells infected with viruses containing a temperature-sensitive defect in the src gene. When cells infected with these viruses were grown at the nonpermissive temperature, more than 90% of the pp60src molecules were associated with pp90 and pp50, and little or no dissociation was observed in a 3-h chase period. These results suggest that mutations in the src gene which affect the transforming activity of pp60src also affect the stability of the interaction of pp60src with pp90 and pp50.
Article
Soybeans, Glycine max, synthesize a family of low-molecular-weight heat shock (HS) proteins in response to HS. The DNA sequences of two genes encoding 17.5- and 17.6-kilodalton HS proteins were determined. Nuclease S1 mapping of the corresponding mRNA indicated multiple start termini at the 5' end and multiple stop termini at the 3' end. These two genes were compared with two other soybean HS genes of similar size. A comparison among the 5' flanking regions encompassing the presumptive HS promoter of the soybean HS-protein genes demonstrated this region to be extremely homologous. Analysis of the DNA sequences in the 5' flanking regions of the soybean genes with the corresponding regions of Drosophila melanogaster HS-protein genes revealed striking similarity between plants and animals in the presumptive promoter structure of thermoinducible genes. Sequences related to the Drosophila HS consensus regulatory element were found 57 to 62 base pairs 5' to the start of transcription in addition to secondary HS consensus elements located further upstream. Comparative analysis of the deduced amino acid sequences of four soybean HS proteins illustrated that these proteins were greater than 90% homologous. Comparison of the amino acid sequence for soybean HS proteins with other organisms showed much lower homology (less than 20%). Hydropathy profiles for Drosophila, Xenopus, Caenorhabditis elegans, and G. max HS proteins showed a similarity of major hydrophilic and hydrophobic regions, which suggests conservation of functional domains for these proteins among widely dispersed organisms.
Article
The products of protein synthesis from exponential phase cultures of Saccharomyces cerevisiae grown at 23 °C or at 36 °C appear to be essentially identical. However, yeast cells respond to a shift in culture temperature from 23 °C to 36 °C with the rapid de novo synthesis of a polypeptide species of molecular weight 100,000. Within 60-90 min after the shift this polypeptide represents approximately 2.5% of the total cellular protein, a 5-10 fold increase over the preshift level. The level of this polypeptide then decreases with continued growth of the cells at 36 °C. Analyses by SDS-polyacrylamide gel electrophoresis of polypeptides obtained from cells pulse labeled with [(35)S]methionine demonstrate that following a temperature shift from 23 °C to 36 °C the synthetic rate of the 100,000 molecular weight polypeptide (as well as a number of other polypeptide species) increases to a level at least 10 fold higher than that observed prior to the shift. A concomittant decrease is observed in the synthesis of a large number of polypeptide species which were actively synthesized before the shift. Maximum changes in synthetic rates are observed 20-30 min after the shift and preshift synthetic patterns are regained within 60-90 min. Synthetic changes of the same magnitude and time course can be produced by short (20-30 min) exposures to 36 °C implicating a heat shock response. Several of the transiently induced polypeptides, including the 100,000 molecular weight species, show an affinity for DNA as determined by DNA-cellulose chromatography.
Article
Synthetic activities at the puff level in salivary gland chromosomes of Drosophila busckii were studied by autoradiographic methods. The analyses were carried out both on normally occurring and on experimentally induced puffs. All the puffs in this material appear to be sites of intense RNA synthesis. When pulse labeling with 3H-thymidine is done to test DNA synthesis in the salivary gland cells, some chromosomes appear continuously labeled along their length, while others are discontinuously labeled. The discontinuously labeled chromosomes are shown to be a phase in the DNA duplication. DNA and RNA synthesis certainly are not mutually exclusive within the entire nucleus and probably not within an individual puff.
Article
The transient activation of heat-shock genes is mediated by a transcription factor which, after heat-shock activation, binds tightly to a regulatory sequence element in their promoters. It is suggested that the developmental activation which acts on some heat-shock genes uses the same activation system, but modifies its activity with additional cell-type-specific factors.
Article
The isolation and partial characterization of two cloned segments of Drosophila melanogaster DNA containing "heat shock" gene sequences is described. We have inserted sheared embryonic D. melanogaster DNA by the poly(dA-dt) connector method (Lobban and Kaiser, 1973) into the R1 restriction site of the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975). A collection of independent hybrid plasmids was screened by colony hybridization (Grunstein and Hogness, 1975) for sequences complementary to in vitro labeled polysomal poly(A)+ heat shock RNA. Two clones were identified which contain sequences complementary to a heat shock mRNA species that directs the in vitro synthesis of the 70,000 dalton heat-induced polypeptide. Both cloned segments hybridize in situ to the heat-induced puff sites located at 87A and 87C of the salivary gland polytene chromosomes.
Article
Three cloned segments of Drosophila melanogaster DNA have been isolated that derive from the major heat shock site at 87C in chromosome 3. Each of these segments contains sequences homologous to a class of polysomal poly(A)+ RNAs whose synthesis is induced by heat shock of cultured cells. A combination of R loop, heteroduplex and restriction fragment maps of these segments reveals that their RNA-homologous sequences are arranged in tandemly repeated units, each unit consisting of an alpha element (0.49 kb) joined to a beta element (1.10 kb). The polysomal RNAs homologous to these alphabeta units (1.59 kb) are distributed into three size classes exhibiting approximate lengths of 1, 2 and 3 kb. R loop mapping demonstrated that the sequence of the 2 kb RNA is alphabetaalpha, indicating that it, and presumably the 3 kb RNA, derive from transcripts covering more than one repeated unit. One of the cloned segments contains a variant repeat unit, alphagamma, located between two alphabeta units. This unit has the same alpha element, but the beta element has been replaced by a nonhomologous gamma element (0.87 kb). Analysis of the total D. melanogaster DNA indicates that the 87C locus contains at least 21 tandemly repeated units, twelve of which were identified as alphabeta units and six as alphagamma units. The 21 or more units are distributed among at least three different tandem arrays separated by different spacer regions, one of which is within a cloned segment. Sequences in the gamma element, but not those in the alpha or beta elements, are also found at 87A, which contains another heat shock site. The possible roles of the alphabeta-type RNAs and of the gamma sequences are discussed in the light of these results.
Article
Two hybrid plasmids, 56H8 and 132E3, which contain D. melanogaster (Dm) DNA segments coding for the 70,000 dalton heat shock protein, have been isolated (Schedl et al., 1978). The analysis of the sequence arrangement presented in the accompanying paper (Moran et al., 1979) shows that both Dm segments contain homologous regions composed of three distinct sequence elements which together define a common unit. We report here that the sequences complementary to the 70,000 dalton protein mRNA appear to be confined to a major portion of the largest element of the common unit and that the other sequence elements are located at the 5' end of the gene. We have also determined the cytogenetic location of the 70,000 dalton protein genes and have investigated whether these sites are transcriptionally active in salivary gland chromosomes.
Article
The isolation of the two hybrid plasmids 56H8 and 132E3, which contain D. melanogaster (Dm) DNA sequences complementary to the mRNA coding for the 70,000 dalton heat shock protein, has been reported (Schedl et al., 1978). Here we compare the sequence arrangement in the two cloned Dm DNA segments by restriction, cross-hybridization and heteroduplex analysis. The results show that the two cloned DNA segments derive from nonoverlapping regions of the Dm genome; that they contain homologous regions present once in 56H8 and twice in 132E3; and that each homologous region is composed of three distinct contiguous sequence elements, x, y and z, which together define a 3 kb common unit. While the 2.5 kb z elements show a high degree of sequence homology in all three common units, the three x and y elements display an intriguing relationship. The localization of the mRNA coding sequences within each of these common units is presented in the accompanying paper (Artavanis-Tsakonas et al., 1979).
Article
When the chromatin of Drosophila is examined by digestion with DNAase I or micrococcal nuclease, no general structural organization above the level of the nucleosome is revealed by the cleavage pattern. In contrast, the DNAase I cleavage pattern of specific regions of the Drosophila chromosome shows discrete bands with sizes ranging from a few kilobase pairs (kb) to more than 20 kb. Visualization of such higher order bands was achieved by the use of the Southern blotting technique. The DNAase I-cleaved fragments were transferred onto a nitrocellulose sheet after size fractionation by gel electrophoresis. Hybridization was then carried out with radioactively labeled cloned fragments of DNA from D. melanogaster. For the five different chromosomal regions examined, each gives a unique pattern of higher order bands on the autoradiogram; the patterns are different for different regions. Restriction enzyme cleavage of the fragments generated indicates that the preferential DNAase I cleavage sites in chromatin are position-specific. The chromosomal regions bounded by preferential DNAase I cleavage sites are referred to as supranucleosomal or higher order domains for purposes of discussion and analysis. The micrococcal nuclease cleavage pattern of chromatin at specific loci was also examined. In the one case studied in detail, this nuclease also cleaves at position-specific sites.
Article
The synthesis and stability of low molecular weight RNAs following heat shock in Drosophilia melanogaster cell cultures have been examined. When cultures are raised from 25 degrees C to 37 degrees C, the synthesis of tRNA and at least two other low molecular weight RNAs continues at the 25 degree C rate. 5.8S ribosomal RNA and most of the low molecular weight nuclear RNAs are not synthesized. The synthesis of 5S ribosomal RNA is greatly reduced. A large amount of an RNA of about 135 nucleotides in length accumulates at 37 degrees C. Nucleotide sequence analysis reveals that this RNA is a novel form of 5S RNA with approximately 15 additional nucleotides at its 3' end.
Article
Messenger RNA transcribed in cultured Drosophila cells adapted for growth under conditions permitting labeling to high specific acitivty has been analyzed by the technique of in situ hybridization. Poly(A)-containing cytoplasmic RNA binds specifically and reproducibly to about 50 bands in the salivary gland polytene chromosomes. In addition heavy labeling of the beta-heterochromatin associated with each of the chromosome arms is observed. The species which are detected probably belong to the more abundant classes of RNA. When the cultured Drosophila cells are subjected to heat shock immediately before labeling with 3H-uridine, there is a drastic alteration in the pattern of gene transcription detected by in situ hybridization. Most of the mRNA synthesis which could be detected in the normal cell is shut off. Newly synthesized RNA hybridizes strongly to seven new sites which do not bind mRNA from control cells. The new loci correspond almost exactly to the regions of Drosophila polytene chromosomes which puff when intact larvae are subjected to an identical heat treatment.
Article
Cytoplasmic 19 S particles were isolated from postpolysomal supernatants of 25 degrees C Drosophila embryos and culture cells. The particles were purified by salt extraction and sucrose gradient centrifugation. Electron microscopic investigation showed that the 19 S particles possess a ring-shaped morphology with an outer diameter of 12 nm and a hollow core of 3 nm. Biochemically the particles are characterized by a group of 16 polypeptides within the molecular weight range of 35 to 23 kDa, and small RNA molecules in the size range of 200 to 60 nucleotides. The RNP character of the particles is also shown by their buoyant density in Cs2SO4 of rho = 1.29 g/cm3 and their susceptibility to uv crosslinking and density in CsCl of rho = 1.38 g/cm3. Antibodies were raised against the proteins of the 19 S particles isolated from 25 degrees C cells and tested by immunoblotting after one- and two-dimensional gel-electrophoresis. Two of the antibodies raised cross react with the small heat-shock proteins hsp 28/27 and hsp 23. Comparative protease V8 cleavage of hsp 23 and the 23-kDa particle protein demonstrates that these two proteins are identical and that the small hsp of Drosophila must be a genuine part of the 19 S cytoplasmic ring-shaped complexes at normal growth temperature. The data support the idea of a general developmental role of some of the so-called heat-shock proteins.
Article
One of the effects of a temperature increase above 35 degrees C on Drosophila melanogaster is a rapid switch in selectivity of the translational apparatus. Protein synthesis from normal, but not from heat shock, mRNA is much reduced. Efficient translation at high temperature might be a result of the primary sequence of heat shock genes. Alternatively a mRNA modification mechanism, altered as a consequence of heat shock, might allow for efficient high temperature translation of any mRNA synthesized during a heat shock. The gene for alcohol dehydrogenase (Adh) was fused to the controlling elements of a heat shock protein 70 (hsp70) gene. Authentic Adh mRNA, synthesized from this fusion gene at elevated temperatures was not translated during heat shock. A second Adh fusion gene in which the mRNA synthesized contained the first 95 nucleotides of the Hsp70 non-translated leader sequence gave rise, at high temperature, to mRNA which was translated during the heat shock. Thus, the signal(s) in the mRNAs controlling translation efficiency at heat shock temperatures is encoded within the heat shock genes.
Article
The present study extends our previous observation (Kasambalides and Lanks, J. Cell. Physiol., 114:93-98, 1983), that dexamethasone inhibits the alterations in heat shock protein (HSP) and glucose-regulated protein (GRP) synthesis caused by glucose deprivation. We now show that insulin, even in the presence of high extracellular glucose concentrations, will induce 95K and 82K GRP synthesis while suppressing 85K and 69K HSP synthesis. Heat shock of insulin-treated cultures causes induction of the 82K GRP rather than the 85K and 69K HSP's. All of the insulin effects are antagonized by dexamethasone. These data suggest that the changes in GRP and HSP synthesis induced by glucose deprivation and heat shock, respectively, may reflect the operation of a normal physiological mechanism that regulates glucose metabolism.
Article
S. typhimurium become resistant to killing by hydrogen peroxide and other oxidants when pretreated with nonlethal levels of hydrogen peroxide. During adaptation to hydrogen peroxide, 30 proteins are induced. Nine are constitutively overexpressed in dominant hydrogen peroxide-resistant oxyR mutants. Mutant oxyR1 is resistant to a variety of oxidizing agents and overexpresses at least five enzyme activities involved in defenses against oxidative damage. Deletions of oxyR are recessive and uninducible by hydrogen peroxide for the nine proteins overexpressed in oxyR1, demonstrating that oxyR is a positive regulatory element. The oxyR1 mutant is also more resistant than the wild-type parent to killing by heat, and it constitutively overexpresses three heat-shock proteins. The oxyR regulatory network is a previously uncharacterized global regulatory system in enteric bacteria.
Article
The ability to identify and purify trans-acting cellular factors that regulate eukaryotic genes is limited by the lack of a practical general assay. Current procedures using crude whole cell or nuclear extracts that restore transcriptional function in vitro or permit reconstruction of native chromatin at control sequences are effective only in select systems. I now present an exonuclease protection assay that is generally applicable for detecting sequence-specific DNA-binding proteins. The assay extends earlier work on the binding to the Drosophila heat-shock gene control element of a protein factor (HAP) present in crude nuclear extracts; the binding was shown by reconstitution of specific exonuclease resistance within a nuclease-hypersensitive site in chromatin. We show here that this same exonuclease resistance can be reconstituted on free linear DNA, despite many nonspecific binding activities present in unfractionated nuclear extracts. We have further applied this assay method to fractionate the protein factor that is bound constitutively to the heat-shock gene TATA box region in native chromatin. Exonuclease protection offers a sensitive, precise and rapid assay for any sequence-specific DNA-binding protein.
Article
The transcriptional regulation of the Drosophila melanogaster hsp27 (also called hsp28) gene was studied by introducing altered genes into the germ line by P element-mediated transformation. DNA sequences upstream of the gene were defined with respect to their effect on steroid hormone-induced and heat-induced transcription. These two types of control were found to be separable; the sequences responsible for 80% of heat-induced expression were located more than 1.1 kilobases upstream of the RNA initiation site, while the sequences responsible for the majority of ecdysterone induction were positioned downstream of the site at -227 base pairs. We have determined the DNA sequence of the intergenic region separating hsp23 and hsp27 and have located putative heat shock and ecdysterone consensus sequences. Our results indicate that the heat shock promoter of the hsp27 gene is organized quite differently from that of hsp70.
Article
At elevated temperatures, germinating conidiospores of Neurospora crassa discontinue synthesis of most proteins and initiate synthesis of three dominant heat shock proteins of 98,000, 83,000, and 67,000 Mr and one minor heat shock protein of 30,000 Mr. Postemergent spores produce, in addition to these, a fourth major heat shock protein of 38,000 Mr and a minor heat shock protein of 34,000 Mr. The three heat shock proteins of lower molecular weight are associated with mitochondria. This exclusive synthesis of heat shock proteins is transient, and after 60 min of exposure to high temperatures, restoration of the normal pattern of protein synthesis is initiated. Despite the transiency of the heat shock response, spores incubated continuously at 45 degrees C germinate very slowly and do not grow beyond the formation of a germ tube. The temperature optimum for heat shock protein synthesis is 45 degrees C, but spores incubated at other temperatures from 40 through 47 degrees C synthesize heat shock proteins at lower rates. Survival was high for germinating spores exposed to temperatures up to 47 degrees C, but viability declined markedly at higher temperatures. Germinating spores survived exposure to the lethal temperature of 50 degrees C when they had been preexposed to 45 degrees C; this thermal protection depends on the synthesis of heat shock proteins, since protection was abolished by cycloheximide. During the heat shock response mitochondria also discontinue normal protein synthesis; synthesis of the mitochondria-encoded subunits of cytochrome c oxidase was as depressed as that of the nucleus-encoded subunits.
Article
Amino acid analogs have been shown to induce heat shock proteins (HSPs). We have examined the effect of these analogs on the thermal sensitivity of Chinese hamster fibroblasts (HA-1) and their stable heat-resistant variants. We found that exposure of HA-1 cells and their heat-resistant variants to canavanine or L-azetidine-2-carboxylic acid cause enhanced synthesis of the three major mammalian HSPs (molecular weight 70,000, 87,000, and 110,000 kd). Although the synthesis of HSPs was increased, the analogs did not induce thermotolerance, a transient ability to protect cells from thermal damage. On the contrary, the analog treatment increased the thermal sensitivity of HA-1 cells, but not of the heat-resistant strains, when these cells were exposed subsequently to elevated temperatures. Our tentative explanation for these findings is that the incorporation of amino acid analogs into HSPs or other cellular proteins sensitizes HA-1 cells to heat. The heat-resistant strains contain higher levels of constitutive HSPs. The additional functional HSPs in the heat-resistant variants may protect these cells from thermal stress. The presence of some newly synthesized analog-substituted, perhaps nonfunctional, HSPs need not affect this thermal protection.
Article
The ability to synthesize a 68,000- to 70,000-Da protein (hsp) in heat-shocked early Xenopus laevis embryos is dependent on the stage of development. Whereas late blastula and later stage embryos synthesize hsp68-70 after heat shock, cleavage stages are incompetent with respect to hsp synthesis. In vitro translation experiments and RNA blot analyses demonstrate that enhanced synthesis of hsp68-70 is associated with an accumulation of hsp68-70 mRNA. Examination of the effect of heat shock on preexisting actin mRNA reveals that heat shock promotes a reduction in the levels of actin mRNA in cleavage embryos but has no discernible effect on actin mRNA levels in neurula embryos. Finally, the acquisition of the heat-shock response (i.e., synthesis of hsp68-70 and accumulation of hsp70 mRNA) during early Xenopus development is correlated with the acquisition of thermotolerance.
Article
Mammalian cells show a complex series of transcriptional and translational switching events in response to heat shock treatment which ultimately lead to the production and accumulation of a small number of proteins, the so-called heat shock (or stress) proteins. We investigated the heat shock response in both qualitative and quantitative ways in cells that were pretreated with drugs that specifically disrupt one or more of the three major cytoskeletal networks. (These drugs alone, cytochalasin E and colcemid, do not result in induction of the heat shock response.) Our results indicated that disruption of the actin microfilaments, the vimentin-containing intermediate filaments, or the microtubules in living cells does not hinder the ability of the cell to undergo an apparently normal heat shock response. Even when all three networks were simultaneously disrupted (resulting in a loose, baglike appearance of the cells), the cells still underwent a complete heat shock response as assayed by the appearance of the heat shock proteins. In addition, the major induced 72-kilodalton heat shock protein was efficiently translocated from the cytoplasm into its proper location in the nucleus and nucleolus irrespective of the condition of the three cytoskeletal elements.
Article
When Drosophila cells are heat shocked, the translation of normal cellular mRNAs is repressed, while mRNAs encoding the heat-shock proteins are translated at high rates. We have found that the hsp70 message is not translated at high temperatures when its leader sequence is deleted. This message is translated when the cells are allowed to recover at 25 degrees C, but the translation ceases when the cells are given a second heat shock. A message with an extra 39 bases added onto the 5' end of the leader behaves in the same way. However, if either of two conserved sequence elements in the leader is deleted, the message is still translated during heat shock. Although the specific feature responsible for the preferential translation of heat-shock messages is not yet identified, we conclude that it must reside in the 5' untranslated leader.
Article
We describe two mutants (tabB-212 and tabB-127) of Escherichia coli K12 in which T-even phage production is temperature-sensitive. Both mutants are linked to purA and may identify a single new bacterial gene tabB. The uninfected bacterium is indistinguishable from wild type at both 30 °C and 42.4 °C. Sodium dodecyl sulphate—polyacrylamide gel electrophoresis of labelled extracts of tabB mutants infected by T4 wild-type phage shows that the modification of viral head precursors (Laemmli, 1970) does not occur, indicating that capsid formation is blocked. The effect is reversible with at least one of the tabB mutants: a shift to 30 °C leads to the cleavage of a significant fraction of precursors synthesized at 42.4 °C.Two classes of T4 mutants are described: one (comB) which grows on tabB even at 42.4 °C, the other (kB) which fails to grow on tabB even at the permissive temperature. Both mutants map in T4 gene 31, suggesting an interaction between gene 31 and tabB products.Since gene 31 mutants lead to the random aggregation of head precursors (Laemmli, 1970), we argue that a host product is involved in the ordered polymerization of T4 proteins into capsids or capsid-related structures.
Article
Mutants of Escherichia coli, called groE, specifically block assembly of bacteriophage λ heads. When groE bacteria are infected by wild type λ, phage adsorption, DNA injection and replication, tail assembly, and cell lysis are all normal. No active heads are formed, however, and head related “monsters” are seen in lysates. These monsters are similar to the structures seen on infection of wild-type cells by phage defective in genes B or C.We have isolated mutants of λ which can overcome the block in groE hosts and have mapped these mutants. All groE mutations can be compensated for by mutation of phage gene E (hence the name groE). Gene E codes for the major structural subunit of the phage head. Some groE mutants, called groEB, can be compensated by mutation in either gene E or in gene B. Gene B is another head gene.During normal head assembly the protein encoded by phage head gene B or C appears to be converted to a lower molecular weight form, h3, which is found in phage. The appearance of h3 protein in fast sedimenting head related structures requires the host groE function.We suggest that the proteins encoded by phage genes E, B and C, and the bacterial component defined by groE mutations act together at an early stage in head assembly.
Article
Electrophoresis studies showed that at least three phage-specified proteins undergo proteolytic cleavage during the development of bacteriophage T5. One of these proteins has a molecular weight of about 135,000 and the product of this cleavage reaction is a minor component of the T5 tail, having a molecular weight of about 128,000. All of the tail-defective T5 mutants studied in this report failed to induce this cleavage reaction under restrictive conditions. This reaction also failed to occur in Escherichia coli groEA639 and groEA36 infected with wild type T5. Examination of lysates of infected groE cells in the electron microscope revealed the presence of filled and empty heads as well as tubular head structures, but no tails were detected. The filled heads were able to combine with separately prepared T5 tails in vitro to form infectious phage particles. Therefore, propagation of T5 in these groE mutants is prevented primarily by a specific block in tail assembly. A T5 mutant, T5ϵ6, was isolated, which has the capacity to propagate in these groE hosts. The gene locus in T5ϵ6 was mapped.The second T5 protein which is cleaved has a molecular weight of 50,000 and is related to head morphogenesis. Treatment of infected cells with l-canavanine (50 μg/ml) inhibited cleavage of this polypeptide. Only small quantities of the major head protein (32,000 mol. wt) were produced in these treated cells. Treatment with canavanine lead to production of tubular heads. The major protein component of partially purified tubular heads has a molecular weight of 50,000. Cells infected with T5 amber H30b, a mutant defective in head gene D20, does not produce the 50,000 and 32,000 molecular weight proteins. These findings suggest that the 50,000 molecular weight protein undergoes cleavage to form the major head polypeptide. A third T5 protein is cleaved to form a minor head component with a molecular weight of 43,000 and its cleavage is linked to that involving the major head protein.
Article
In order to separate some of the factors involved in the formation of puffs the antibiotic actinomycin D was applied at different stages of puff activity. Puffs were induced by temperature shocks or eodysone. Inhibition of RNA synthesis with actinomycin D before application of a puff inducing stimulus prevents neither the appearance of the stimulus specific puffs nor the accumulation of acidic proteins in the puff regions. The puffs attained under these conditions approximately 1/3 of the size normally produced by the stimulus. Indications were obtained that during puff formation acidic protein accumulation precedes the onset of RNA synthesis. Synthesis and storage of newly synthesized RNA within the puff region was studied on the basis of grain distribution in uridine-H3 autoradiographs after various incubation periods. RNA synthesis appears to be restricted to a particular area of the puff region. After a 3 min temperature shock following injection of uridine-H3 silver grains are located only over a particular area of the newly formed puff. The same area becomes labeled during a 1 min pulse of uridine-H3 applied at a stage of maximum puff development. Longer periods of incubation result in a random distribution of the grains over the whole puff region. Grain counts on different areas of experimentally induced puffs and on the same areas at a stage of puff regression indicate that the newly synthesized RNA becomes transferred from the area where it was synthesized and is stored for a certain period within the puff region. Complete release of newly synthesized RNA from puffs in which RNA synthesis was inhibited by actinomycin D at a stage of maximal activity is accomplished within 30 to 35 min.