Stress response gene family expansions correlate with invasive potential in teleost fish.

The bluegill sunfish Lepomis macrochirus and the closely related redear sunfish Lepomis microlophus have important ecological and recreational value and are widely used for research and aquaculture. While both species have been introduced outside of their native ranges, only the bluegill is considered invasive. Here, we report de novo transcriptome assemblies for these fish as a resource for sunfish biology. Comparative analyses of the transcriptomes revealed an unexpected, bluegill-specific expansion in the HSP70 and HSP90 molecular chaperone gene families. These expansions were not unique to the bluegill as expansions in HSP70s and HSP90s were identified in the genomes of other teleost fish using the NCBI RefSeq database. To determine whether gene family expansions are specific for thermal stress responses, GST and SOD gene families that are associated with oxidative stress responses were also analyzed. Species-specific expansions were also observed for these gene families in distinct fish species. Validating our approach, previously described expansions in the MHC gene family were also identified. Intriguingly, the number of HSP70 paralogs was positively correlated with thermotolerance range for each species, suggesting that these expansions can impact organismal physiology. Furthermore, fish that are considered invasive contained a higher average number of HSP70 paralogs than non-invasive fish. Invasive fish also had higher average numbers of HSP90, MHC and GST paralogs, but not SOD paralogs. Taken together, we propose that expansions in key cellular stress response gene families represent novel genetic signatures that correlate with invasive potential.

Chronic temperature stress inhibits reproduction and disrupts endocytosis via chaperone titration in Caenorhabditis elegans.

BACKGROUND: Temperature influences biology at all levels, from altering rates of biochemical reactions to determining sustainability of entire ecosystems. Although extended exposure to elevated temperatures influences organismal phenotypes important for human health, agriculture, and ecology, the molecular mechanisms that drive these responses remain largely unexplored. Prolonged, mild temperature stress (48 h at 28 °C) has been shown to inhibit reproduction in Caenorhabditis elegans without significantly impacting motility or viability. RESULTS: Analysis of molecular responses to chronic stress using RNA-seq uncovers dramatic effects on the transcriptome that are fundamentally distinct from the well-characterized, acute heat shock response (HSR). While a large portion of the genome is differentially expressed ≥ 4-fold after 48 h at 28 °C, the only major class of oogenesis-associated genes affected is the vitellogenin gene family that encodes for yolk proteins (YPs). Whereas YP mRNAs decrease, the proteins accumulate and mislocalize in the pseudocoelomic space as early as 6 h, well before reproduction declines. A trafficking defect in a second, unrelated fluorescent reporter and a decrease in pre-synaptic neuronal signaling indicate that the YP mislocalization is caused by a generalized defect in endocytosis. Molecular chaperones are involved in both endocytosis and refolding damaged proteins. Decreasing levels of the major HSP70 chaperone, HSP-1, causes similar YP trafficking defects in the absence of stress. Conversely, increasing chaperone levels through overexpression of the transcription factor HSF-1 rescues YP trafficking and restores neuronal signaling. CONCLUSIONS: These data implicate chaperone titration during chronic stress as a molecular mechanism contributing to endocytic defects that influence multiple aspects of organismal physiology. Notably, HSF-1 overexpression improves recovery of viable offspring after exposure to stress. These findings provide important molecular insights into understanding organismal responses to temperature stress as well as phenotypes associated with chronic protein misfolding.

Synthesis and Characterization of Ligand-Stabilized Silver Nanoparticles and Comparative Antibacterial Activity against E. coli.

Silver is a well-established antimicrobial agent. Conjugation of organic ligands with silver nanoparticles has been shown to create antimicrobial nanoparticles with improved pharmacodynamic properties and reduced toxicity. Twelve novel organic ligand functionalized silver nanoparticles (AgNPs) were prepared via a light-controlled reaction with derivatives of benzothiazole, benzoxazine, quinazolinone, 2-butyne-1,4-diol, 3-butyne-1-ol, and heptane-1,7-dioic. UV-vis, Fourier-transform infrared (FTIR) spectroscopy, and energy-dispersive X-ray (EDAX) analysis were used to confirm the successful formation of ligand-functionalized nanoparticles. Dynamic light scattering (DLS) revealed mean nanoparticle diameters between 25 and 278 nm. Spherical and nanotube-like morphologies were observed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Seven of the twelve nanoparticles exhibited strong antimicrobial activity and five of the twelve demonstrated significant antibacterial capabilities against E. coli in a zone-of-inhibition assay. The synthesis of functionalized silver nanoparticles such as the twelve presented is critical for the further development of silver-nanoconjugated antibacterial agents.

Titration of SF3B1 Activity Reveals Distinct Effects on the Transcriptome and Cell Physiology.

SF3B1 is a core component of the U2 spliceosome that is frequently mutated in cancer. We have previously shown that titrating the activity of SF3B1, using the inhibitor pladienolide B (PB), affects distinct steps of the heat shock response (HSR). Here, we identify other genes that are sensitive to different levels of SF3B1 (5 vs. 100 nM PB) using RNA sequencing. Significant changes to mRNA splicing were identified at both low PB and high PB concentrations. Changes in expression were also identified in the absence of alternative splicing, suggesting that SF3B1 influences other gene expression pathways. Surprisingly, gene expression changes identified in low PB are not predictive of changes in high PB. Specific pathways were identified with differential sensitivity to PB concentration, including nonsense-mediated decay and protein-folding homeostasis, both of which were validated using independent reporter constructs. Strikingly, cells exposed to low PB displayed enhanced protein-folding capacity relative to untreated cells. These data reveal that the transcriptome is exquisitely sensitive to SF3B1 and suggests that the activity of SF3B1 is finely regulated to coordinate mRNA splicing, gene expression and cellular physiology.

Caged Proline in Photoinitiated Organocatalysis.

Organocatalysis is an emerging field, in which small metal-free organic structures catalyze a diversity of reactions with a remarkable stereoselectivity. The ability to selectively switch on such pathways upon demand has proven to be a valuable tool in biological systems. Light as a trigger provides the ultimate spatial and temporal control of activation. However, there have been limited examples of phototriggered catalytic systems. Herein, we describe the synthesis and application of a caged proline system that can initiate organocatalysis upon irradiation. The caged proline was generated using the highly efficient 4-carboxy-5,7-dinitroindolinyl (CDNI) photocleavable protecting group in a four-step synthesis. Advantages of this system include water solubility, biocompatibility, high quantum yield for catalyst release, and responsiveness to two-photon excitation. We showed the light-triggered catalysis of a crossed aldol reaction, a Mannich reaction, and a self-aldol condensation reaction. We also demonstrated light-initiated catalysis, leading to the formation of a biocide in situ, which resulted in the growth inhibition of E. coli, with as little as 3 min of irradiation. This technique can be broadly applied to other systems, by which the formation of active forms of drugs can be catalytically assembled remotely via two-photon irradiation.

Identification of a tissue-selective heat shock response regulatory network.

The heat shock response (HSR) is essential to survive acute proteotoxic stress and has been studied extensively in unicellular organisms and tissue culture cells, but to a lesser extent in intact metazoan animals. To identify the regulatory pathways that control the HSR in Caenorhabditis elegans, we performed a genome-wide RNAi screen and identified 59 genes corresponding to 7 positive activators required for the HSR and 52 negative regulators whose knockdown leads to constitutive activation of the HSR. These modifiers function in specific steps of gene expression, protein synthesis, protein folding, trafficking, and protein clearance, and comprise the metazoan heat shock regulatory network (HSN). Whereas the positive regulators function in all tissues of C. elegans, nearly all of the negative regulators exhibited tissue-selective effects. Knockdown of the subunits of the proteasome strongly induces HS reporter expression only in the intestine and spermatheca but not in muscle cells, while knockdown of subunits of the TRiC/CCT chaperonin induces HS reporter expression only in muscle cells. Yet, both the proteasome and TRiC/CCT chaperonin are ubiquitously expressed and are required for clearance and folding in all tissues. We propose that the HSN identifies a key subset of the proteostasis machinery that regulates the HSR according to the unique functional requirements of each tissue.

Visualization of the biphasic calcium wave during fertilization in Caenorhabditis elegans using a genetically encoded calcium indicator.

Fertilization is a critical step in development, yet internal fertilization events are notoriously difficult to visualize. Taking advantage of the calcium response that is a hallmark of sperm-egg fusion, we adapted the genetically encoded calcium indicator jGCaMP7s to visualize the moment of fertilization in Caenorhabditis elegans using fluorescence. We termed this tool the 'CaFE' reporter, for 'calcium during fertilization in C. elegans'. The CaFE reporter produced a robust signal that recapitulated the previously reported, biphasic nature of the calcium wave and had no significant deleterious effects on worm physiology or fecundity. Calcium waves were not observed at the restrictive temperature in the spe-9(hc88) strain, in which sperm can still trigger meiotic maturation but can no longer fuse with the oocyte. Demonstrating the utility of the CaFE reporter, we analyzed polyspermy induced by inhibition of egg-3 via RNAi and observed late calcium waves in the uterus. This finding provides support to the idea that calcium release is not restricted to the first sperm fusion event during polyspermy. Establishment of the CaFE reporter in the genetically tractable and optically transparent worm provides a powerful tool to dissect the oocyte-to-embryo transition inside a living animal.

Praziquantel inhibits Caenorhabditis elegans development and species-wide differences might be cct-8-dependent.

Anthelmintic drugs are used to treat parasitic roundworm and flatworm infections in humans and other animals. Caenorhabditis elegans is an established model to investigate anthelmintics used to treat roundworms. In this study, we use C. elegans to examine the mode of action and the mechanisms of resistance against the flatworm anthelmintic drug praziquantel (PZQ), used to treat trematode and cestode infections. We found that PZQ inhibited development and that this developmental delay varies by genetic background. Interestingly, both enantiomers of PZQ are equally effective against C. elegans, but the right-handed PZQ (R-PZQ) is most effective against schistosome infections. We conducted a genome-wide association mapping with 74 wild C. elegans strains to identify a region on chromosome IV that is correlated with differential PZQ susceptibility. Five candidate genes in this region: cct-8, znf-782, Y104H12D.4, Y104H12D.2, and cox-18, might underlie this variation. The gene cct-8, a subunit of the protein folding complex TRiC, has variation that causes a putative protein coding change (G226V), which is correlated with reduced developmental delay. Gene expression analysis suggests that this variant correlates with slightly increased expression of both cct-8 and hsp-70. Acute exposure to PZQ caused increased expression of hsp-70, indicating that altered TRiC function might be involved in PZQ responses. To test if this variant affects development upon exposure to PZQ, we used CRISPR-Cas9 genome editing to introduce the V226 allele into the N2 genetic background (G226) and the G226 allele into the JU775 genetic background (V226). These experiments revealed that this variant was not sufficient to explain the effects of PZQ on development. Nevertheless, this study shows that C. elegans can be used to study PZQ mode of action and resistance mechanisms. Additionally, we show that the TRiC complex requires further evaluation for PZQ responses in C. elegans.

Divergent regulatory roles of NuRD chromatin remodeling complex subunits GATAD2 and CHD4 in Caenorhabditis elegans.

During proteotoxic stress, a pathway known as the heat shock response is induced to maintain protein-folding homeostasis or proteostasis. Previously, we identified the Caenorhabditis elegans GATAD2 ortholog, dcp-66, as a novel regulator of the heat shock response. Here, we extend these findings to show that dcp-66 positively regulates the heat shock response at the cellular, molecular, and organismal levels. As GATAD2 is a subunit of the nucleosome remodeling and deacetylase chromatin remodeling complex, we examined other nucleosome remodeling and deacetylase subunits and found that the let-418 (CHD4) nucleosome repositioning core also regulates the heat shock response. However, let-418 acts as a negative regulator of the heat shock response, in contrast to positive regulation by dcp-66. The divergent effects of these two nucleosome remodeling and deacetylase subunits extend to the regulation of other stress responses including oxidative, genotoxic, and endoplasmic reticulum stress. Furthermore, a transcriptomic approach reveals additional divergently regulated pathways, including innate immunity and embryogenesis. Taken together, this work establishes new insights into the role of nucleosome remodeling and deacetylase subunits in organismal physiology. We incorporate these findings into a molecular model whereby different mechanisms of recruitment to promoters can result in the divergent effects of nucleosome remodeling and deacetylase subunits.

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

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

Standardized Methods for Measuring Induction of the Heat Shock Response in Caenorhabditis elegans.

The heat shock response (HSR) is a cellular stress response induced by cytosolic protein misfolding that functions to restore protein folding homeostasis, or proteostasis. Caenorhabditis elegans occupies a unique and powerful niche for HSR research because the HSR can be assessed at the molecular, cellular, and organismal levels. Therefore, changes at the molecular level can be visualized at the cellular level and their impacts on physiology can be quantitated at the organismal level. While assays for measuring the HSR are straightforward, variations in the timing, temperature, and methodology described in the literature make it challenging to compare results across studies. Furthermore, these issues act as a barrier for anyone seeking to incorporate HSR analysis into their research. Here, a series of protocols is presented for measuring induction of the HSR in a robust and reproducible manner with RT-qPCR, fluorescent reporters, and an organismal thermorecovery assay. Additionally, we show that a widely used thermotolerance assay is not dependent on the well-established master regulator of the HSR, HSF-1, and therefore should not be used for HSR research. Finally, variations in these assays found in the literature are discussed and best practices are proposed to help standardize results across the field, ultimately facilitating neurodegenerative disease, aging, and HSR research.

Temperature-induced physiological stress and reproductive characteristics of the migratory seahorse Hippocampus erectus during a thermal stress simulation.

Inshore-offshore migration occurs frequently in seahorse species, either because of prey opportunities or because they are driven by reproduction, and variations in water temperature may dramatically change migratory seahorse behavior and physiology. The present study investigated the behavioral and physiological responses of the lined seahorse Hippocampus erectus under thermal stress and evaluated the potential effects of different temperatures on its reproduction. The results showed that the thermal tolerance of the seahorses was time dependent. Acute thermal stress (30°C, 2-10 h) increased the basal metabolic rate (breathing rate) and the expression of stress response genes (Hsp genes) significantly and further stimulated seahorse appetite. Chronic thermal treatment (30°C, 4 weeks) led to a persistently higher basal metabolic rate, higher stress response gene expression and higher mortality rates, indicating that the seahorses could not acclimate to chronic thermal stress and might experience massive mortality rates due to excessively high basal metabolic rates and stress damage. Additionally, no significant negative effects on gonad development or reproductive endocrine regulation genes were observed in response to chronic thermal stress, suggesting that seahorse reproductive behavior could adapt to higher-temperature conditions during migration and within seahorse breeding grounds. In conclusion, this simulation experiment indicates that temperature variations during inshore-offshore migration have no effect on reproduction, but promote significantly high basal metabolic rates and stress responses. Therefore, we suggest that the observed high tolerance of seahorse reproduction is in line with the inshore-offshore reproductive migration pattern of lined seahorses.This article has an associated First Person interview with the first author of the paper.

SF3B1 is a stress-sensitive splicing factor that regulates both HSF1 concentration and activity.

The heat shock response (HSR) is a well-conserved, cytoprotective stress response that activates the HSF1 transcription factor. During severe stress, cells inhibit mRNA splicing which also serves a cytoprotective function via inhibition of gene expression. Despite their functional interconnectedness, there have not been any previous reports of crosstalk between these two pathways. In a genetic screen, we identified SF3B1, a core component of the U2 snRNP subunit of the spliceosome, as a regulator of the heat shock response in Caenorhabditis elegans. Here, we show that this regulatory connection is conserved in cultured human cells and that there are at least two distinct pathways by which SF3B1 can regulate the HSR. First, inhibition of SF3B1 with moderate levels of Pladienolide B, a previously established small molecule inhibitor of SF3B1, affects the transcriptional activation of HSF1, the transcription factor that mediates the HSR. However, both higher levels of Pladienolide B and SF3B1 siRNA knockdown also change the concentration of HSF1, a form of HSR regulation that has not been previously documented during normal physiology but is observed in some forms of cancer. Intriguingly, mutations in SF3B1 have also been associated with several distinct types of cancer. Finally, we show that regulation of alternative splicing by SF3B1 is sensitive to temperature, providing a new mechanism by which temperature stress can remodel the transcriptome.

Cellular Proteomes Drive Tissue-Specific Regulation of the Heat Shock Response.

The heat shock response (HSR) is a cellular stress response that senses protein misfolding and restores protein folding homeostasis, or proteostasis. We previously identified an HSR regulatory network in Caenorhabditis elegans consisting of highly conserved genes that have important cellular roles in maintaining proteostasis. Unexpectedly, the effects of these genes on the HSR are distinctly tissue-specific. Here, we explore this apparent discrepancy and find that muscle-specific regulation of the HSR by the TRiC/CCT chaperonin is not driven by an enrichment of TRiC/CCT in muscle, but rather by the levels of one of its most abundant substrates, actin. Knockdown of actin subunits reduces induction of the HSR in muscle upon TRiC/CCT knockdown; conversely, overexpression of an actin subunit sensitizes the intestine so that it induces the HSR upon TRiC/CCT knockdown. Similarly, intestine-specific HSR regulation by the signal recognition particle (SRP), a component of the secretory pathway, is driven by the vitellogenins, some of the most abundant secretory proteins. Together, these data indicate that the specific protein folding requirements from the unique cellular proteomes sensitizes each tissue to disruption of distinct subsets of the proteostasis network. These findings are relevant for tissue-specific, HSR-associated human diseases such as cancer and neurodegenerative diseases. Additionally, we characterize organismal phenotypes of actin overexpression including a shortened lifespan, supporting a recent hypothesis that maintenance of the actin cytoskeleton is an important factor for longevity.

Convergence of molecular, modeling, and systems approaches for an understanding of the Escherichia coli heat shock response.

The heat shock response (HSR) is a homeostatic response that maintains the proper protein-folding environment in the cell. This response is universal, and many of its components are well conserved from bacteria to humans. In this review, we focus on the regulation of one of the most well-characterized HSRs, that of Escherichia coli. We show that even for this simple model organism, we still do not fully understand the central component of heat shock regulation, a chaperone-mediated negative feedback loop. In addition, we review other components that contribute to the regulation of the HSR in E. coli and discuss how these additional components contribute to regulation. Finally, we discuss recent genomic experiments that reveal additional functional aspects of the HSR.

Hfq modulates the sigmaE-mediated envelope stress response and the sigma32-mediated cytoplasmic stress response in Escherichia coli.

Hfq, a chaperone for small noncoding RNAs, regulates many processes in Escherichia coli, including the sigma(S)-mediated general stress response. Here we used microarray analysis to identify the changes in gene expression resulting from lack of Hfq. We identify several potential new targets for Hfq regulation, including genes encoding outer membrane proteins, enzymes, factors, and transporters. Many of these genes are involved in amino acid uptake and biosynthesis, sugar uptake and metabolism, and cell energetics. In addition, we find altered regulation of the sigma(E)- and sigma(32)-mediated stress responses, which we analyze further. We show that cells lacking Hfq induce the sigma(E)-mediated envelope stress response and are defective in sigma(E)-mediated repression of outer membrane proteins. We also show that the sigma(32)-mediated cytoplasmic stress response is repressed in cells lacking Hfq due to increased expression of DnaK. Furthermore, we show that cells lacking Hfq are defective in the "long-term adaptation" of sigma(32) to chronic chaperone overexpression. Together, our results indicate that Hfq may play a general role in stress response regulation in E. coli.

Analysis of sigma32 mutants defective in chaperone-mediated feedback control reveals unexpected complexity of the heat shock response.

Protein quality control is accomplished by inducing chaperones and proteases in response to an altered cellular folding state. In Escherichia coli, expression of chaperones and proteases is positively regulated by sigma32. Chaperone-mediated negative feedback control of sigma32 activity allows this transcription factor to sense the cellular folding state. We identified point mutations in sigma32 altered in feedback control. Surprisingly, such mutants are resistant to inhibition by both the DnaK/J and GroEL/S chaperones in vivo and also show dramatically increased stability. Further characterization of the most defective mutant revealed that it has almost normal binding to chaperones and RNA polymerase and is competent for chaperone-mediated inactivation in vitro. We suggest that the mutants identify a regulatory step downstream of chaperone binding that is required for both inactivation and degradation of sigma32.

A chaperone network controls the heat shock response in E. coli.

The heat shock response controls levels of chaperones and proteases to ensure a proper cellular environment for protein folding. In Escherichia coli, this response is mediated by the bacterial-specific transcription factor, sigma32. The DnaK chaperone machine regulates both the amount and activity of sigma32, thereby coupling sigma32 function to the cellular protein folding state. In this manuscript, we analyze the ability of other major chaperones in E. coli to regulate sigma32, and we demonstrate that the GroEL/S chaperonin is an additional regulator of sigma32. We show that increasing the level of GroEL/S leads to a decrease in sigma32 activity in vivo and this effect can be eliminated by co-overexpression of a GroEL/S-specific substrate. We also show that depletion of GroEL/S in vivo leads to up-regulation of sigma32 by increasing the level of sigma32. In addition, we show that changing the levels of GroEL/S during stress conditions leads to measurable changes in the heat shock response. Using purified proteins, we show that that GroEL binds to sigma32 and decreases sigma32-dependent transcription in vitro, suggesting that this regulation is direct. We discuss why using a chaperone network to regulate sigma32 results in a more sensitive and accurate detection of the protein folding environment.