Cell Stress and Chaperones

Heat shock proteins (HSPs) are ubiquitous molecular chaperones that safeguard proteostasis under stress. We first investigated the expression dynamics of the mitochondrial chaperonin HSP60 across diverse cnidarians to understand its stress-responsive regulation. Using immunoblotting, we quantified HSP60 expression in Pocillopora acuta (reef-building coral), Exaiptasia diaphana (sea anemone), and Cassiopea xamachana (upside-down jellyfish). In P. acuta, HSP60 was not detected at the fragment scale under either control or heat stress, whereas isolated cells exhibited transient HSP60 expression during exposure to both control and heated temperatures (+5 {degrees}C above optimum), indicating that HSP60 regulation in this coral is strongly context-dependent and potentially suppressed at the tissue level. In contrast, E. diaphana and C. xamachana showed gradual, and temperature-dependent accumulation of HSP60 over 24 h under heated conditions (+5 {degrees}C above its thermal optimum), however C. xamachana also displayed constitutive basal expression under control conditions. These contrasting profiles highlight clear lineage-specific differences in HSP60 regulation among cnidarians. The consistent antibody cross-reactivity observed across all three species then prompted us to explore the evolutionary basis of this conservation. Phylogenetic analyses of HSP60 sequences confirmed that cnidarian proteins are orthologous to the canonical vertebrate HSP60 (human HSPD1), demonstrating deep structural and evolutionary conservation of this chaperonin across Metazoa. Collectively, these findings reveal that while HSP60 is evolutionarily ancient and conserved, its regulation under thermal stress varies across lineages and physiological context, reflecting complex modulation of mitochondrial proteostasis in early-diverging metazoans. This lineage- and context-dependent regulatory framework provides new insight into how chaperone plasticity contributes to cnidarian thermal tolerance and the differential susceptibility of reef taxa to bleaching under ocean warming. Significance StatementHeat shock protein 60 (HSP60) is a highly conserved mitochondrial chaperonin critical for maintaining protein homeostasis, yet its regulatory dynamics across early-diverging animal lineages are poorly understood. By first comparing the expression responses of three phylogenetically and ecologically distinct cnidarians--the coral Pocillopora acuta, the sea anemone Exaiptasia diaphana, and the upside-down jellyfish Cassiopea xamachana--we uncovered clear lineage-specific differences in HSP60 regulation. P. acuta showed no detectable HSP60 induction in intact tissue, underscoring strong context-dependence that may prevent the deployment of this critical molecular defense mechanism, reflecting its high thermal susceptibility. In contrast, E. diaphana and C. xamachana displayed gradual, temperature-dependent accumulation aligning with their thermal flexibility, with C. xamachana also displaying constitutive basal levels under control condition. The consistent antibody cross-reactivity across all three species then led us to investigate evolutionary conservation, revealing that cnidarian HSP60s are orthologous to the canonical HSP60 (human HSPD1). This demonstrates that HSP60 is deeply conserved from cnidarians to mammals, yet its stress-responsive regulation has diversified across lineages and physiological contexts. This lineage- and context-dependent regulatory framework illuminates how fundamental differences in chaperone control shape cnidarian stress physiology, offering new mechanistic insight into the cellular basis of coral bleaching susceptibility under ocean warming.

Thermal fluctuations in the environment, particularly high temperatures, pose a major challenge to organisms and require robust mechanisms for survival under heat stress. Although the molecular basis of cellular heat shock responses is well understood, how thermosensory neurons contribute to systemic stress adaptation remains unclear. Here, using Caenorhabditis elegans as a model, we examine whether ther-mosensory receptor guanylyl cyclases (rGCs) in AFD neurons regulate organism-wide stress responses under noxious temperatures and how individual rGCs contribute to this coordination. Among the AFD-expressed rGCs, we identify gcy-18 and gcy-23 as key regulators of the physiological response to thermal stress, acting through modulation of canonical heat shock response (HSR) genes. Our findings indicate that rGC signaling is crucial for activation of heat shock chaperones and maintenance of proteostasis under high temperatures (35{degrees}C). Supporting this, quantitative analysis of HSP-16.2 revealed that rGC activity in AFD neurons modulates the HSR magnitude in distal tissues, such as the intestine. Together, our findings uncover an important role for thermosensory rGCs in maintaining cellular proteostasis through selective modulation of the HSR.

Heat shock proteins (HSPs) are well known to prevent and repair protein damage caused by various abiotic stressors, but their role in low temperature and freezing stress is not well-characterized compared to other thermal challenges. Ice formation in and around cells is hypothesized to cause protein damage, yet many species of insects can survive freezing, suggesting HSPs may be an important mechanism in freeze tolerance. Here, we studied HSP70 in a freeze-tolerant cricket Gryllus veletis to better understand the role of HSPs in this phenomenon. We measured expression of one heat-inducible HSP70 isoform at the mRNA level (using RT-qPCR), as well as the relative abundance of total HSP70 protein (using semi-quantitative Western blotting), in five tissues from crickets exposed to a survivable heat treatment (2 h at 40{degrees}C), a 6-week fall-like acclimation that induces freeze tolerance, and a survivable freezing treatment (1.5 h at -8{degrees}C). While HSP70 expression was upregulated by heat at the mRNA or protein level in all tissues studied (fat body, Malphigian tubules, midgut, femur muscle, nervous system ganglia), no tissue exhibited HSP70 upregulation within 2 - 24 h following a survivable freezing stress. During fall-like acclimation to mild low temperatures, we only saw moderate upregulation of HSP70 at the protein level in muscle, and at the RNA level in fat body and nervous tissue. Although HSP70 is important for responding to a wide range of stressors, our work suggests that this chaperone may be less critical in the preparation for, and response to, moderate freezing stress. HighlightsO_LIHeat shock protein 70 (HSP70) may not contribute substantially to freeze tolerance C_LIO_LIHeat stress caused HSP70 mRNA and protein upregulation in the spring field cricket C_LIO_LIAcclimation prior to freezing was correlated with slight HSP70 upregulation C_LIO_LIHSP70 was not upregulated after freezing in this freeze-tolerant insect C_LIO_LIFurther work is needed to determine whether freezing causes protein damage C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/621172v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@170fda7org.highwire.dtl.DTLVardef@11cf432org.highwire.dtl.DTLVardef@1e41be9org.highwire.dtl.DTLVardef@e46c4d_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hibernation consists of bouts of torpor, characterized by profound decreases in metabolism and body temperature (Tb), alternated with periods of euthermia called interbout arousals, during which normal metabolism and Tb resume. Seasonal hibernators accumulate DNA strand breaks during torpor, which are repaired during arousal. Here, we assess dynamics of DNA damage and repair during serial daily torpor in mice induced by 30% calorie restriction (CR) and investigate the effects of metabolic challenge on DNA repair. Serial daily torpor induced by CR in C57/BL6J mice of both sexes housed at 20{degrees}C lasts 6-12 hours. Like seasonal hibernators, DNA damage increases in CR-induced torpor and is repaired in the subsequent euthermic period, as evidenced by comet assay and {gamma}H2AX accumulation. To metabolically challenge animals, ambient temperature (Ta) was lowered to 4{degrees}C, since torpid mice defend a Tb of around 20{degrees}C or higher. Despite inducing a significant metabolic challenge, housing of torpid mice at 4{degrees}C does not increase DNA damage compared to 20{degrees}C housing. However, reducing Ta to 4{degrees}C during euthermia inhibits DNA repair. Interestingly, p21 levels increase in mice exposed to 4{degrees}C, indicating cell-cycle inhibition during exposure to 4{degrees}C. Thus, 30% CR induces daily cycles of torpor-induced DNA damage and euthermia-associated DNA repair in mice, and exposure to a Ta of 4{degrees}C during arousal inhibits DNA repair mounting a cell cycle inhibition response. Thus, the torpor-arousal cycle may be a contributing factor to the lifespan extension benefits of CR in mice, promoting genomic integrity and thereby cellular and tissue health.

The nucleolus is a dynamic nuclear biomolecular condensate involved in cellular stress response. Under proteotoxic stress, the nucleolus can store damaged proteins for refolding or degradation. HSP70 chaperone is a well-documented player in the recovery process of proteins accumulated in the nucleolus after heat shock. However, little is known about the involvement of the ubiquitin-proteasome system in the turnover of its nucleolar clients. Here we show that HSP70, independently of its ATPase activity, promotes migration of the CHIP (carboxyl terminus of HSC70-interacting protein) ubiquitin ligase into the granular component of the nucleolus, specifically after heat stress. We show that while in the nucleolus, CHIP retains mobility that depends on its ubiquitination activity. Furthermore, after prolonged exposure to heat stress, CHIP self-organizes into large, intra-nucleolar droplet-like structures whose size is determined by CHIP ubiquitination capacity. Using a heat-sensitive nucleolar protein luciferase, we show that excess CHIP impairs its regeneration, probably through deregulation of HSP70. Our results demonstrate a novel role for CHIP in managing nucleolar proteostasis in response to stress.

Heatstroke is a life-threatening condition in which heat-shock and unfolded-protein responses are strongly activated but fail to prevent proteostasis disruption and severe cellular injury. Whether post-transcriptional regulation contributes to this mismatch remains unknown. We integrated small RNA sequencing with mRNA profiling in peripheral blood mononuclear cells from patients with classical heatstroke and matched heat-exposed controls recruited during the Hajj pilgrimage. mRNA profiling was performed in 19 cases and 19 controls, and miRNA sequencing in 17 cases and 16 controls from the same cohort. Differentially expressed miRNAs were integrated with 4,462 differentially expressed mRNAs using high-confidence inverse-expression miRNA-mRNA pairs. Twenty-six miRNAs mapped to 376 mRNA targets, forming 414 regulatory pairs and two opposing programmes. Programme A, comprising 16 downregulated miRNAs, was associated with activation of PI3K-mTOR, NRF2 oxidative stress and HIF-1 signalling, consistent with stress-survival signalling. Programme B, comprising 10 upregulated miRNAs, was associated with suppression of stress-granule components and fatty-acid {beta}-oxidation genes, consistent with impaired protein quality control and reduced metabolic flexibility. miR-92a-3p emerged as a central regulatory node, and its target PIK3R3 connected 9 of the 10 enriched pathways. These findings suggest a post-transcriptional regulatory layer that could contribute to the limited protection afforded by activated stress defences in human heatstroke.

Heat shock proteins in parallel with their main and originally discovered function - maintenance of folded proteins under stressful conditions, can play also background buffering role - by folding proteins with an excess of slightly-deleterious nonsynonymous variants (SDNV). Here we tested several scenarios of this buffering role. On the comparative species scale, we demonstrated that low-Ne species are characterized by a higher expression level of hsp90 which can be explained by the excess of SDNV. On the comparative tissue level, we showed that long-lived tissues have also a higher hsp90 expression level, which can be advantageous to maintain the functionality of proteins. On the comparative gene level, we demonstrated that purifying selection of hsp90 in low-Ne-species did not relax as strongly as it happens for control genes, similar to hsp90. Additionally, we demonstrated that hsp clients versus non-clients are characterised by decreased level of selective constraints; demonstrate stronger relaxation of purifying selection in low-Ne species; have an excess of slightly-deleterious variants associated with complex disease phenotypes in humans; have an excess of pathological variants associated with clinical phenotypes in humans, suggesting that clients, being buffered by hsp90 can degenerate a bit more as compared to non-clients. Altogether, our results show that the secondary role of hsp, buffering of SDNV, is widespread and universal affecting properties of species, tissues and genes. A deep understanding of the buffering role of hsp90 will help to predict the deleterious effect of each variant in the human genome more precisely as well as will extend the application of the effectively-neutral theory of molecular evolution.

The heat shock response (HSR), driven by molecular chaperons, is a key defense mechanism against proteotoxic stress. In nematodes, the hsp16 family and hsp70 family play central roles in the HSR, but their evolutionary conservation and expression patterns remain poorly understood. Here, we performed phylogenetic, genomic, and transcriptomic analyses of hsp16 and hsp70 genes in Caenorhabditis elegans and C. briggsae, and other related nematodes. Our findings show that hsp16 genes are rapidly evolving and often organized in clusters of closely spaced and oppositely oriented pairs. These genes show dynamic temporal expression in C. elegans and C. briggsae under basal, unstressed conditions. In contrast, hsp70 genes are more conserved across species and display stable expression. We also found that C. briggsae contains more orthologous HSP genes than C. elegans, and exhibits a higher proportion of constitutive expression, consistent with its greater thermal tolerance. These findings highlight the evolutionary diversification and functional organization of heat shock proteins in nematodes, offering insights into how genomic architecture and gene expression contribute to species-specific thermal adaptation.

The muroid rodent Nannospalax galili (syn. Spalax) is adapted to life in underground burrows and tolerates acute exposure to severe hypoxia. Adaptation to hypoxia is correlated with delayed onset of ageing and resistance against tumour formation. Spalax becomes five to seven times older than its relatives, the mouse and rat, without displaying signs of ageing or developing ageing-related disorders like cancer. Investigating and understanding adapted genes and gene regulatory networks of Spalax might pinpoint novel strategies to maintain an extended healthy phenotype in humans. Here we analysed and compared RNA-Seq data of liver, kidney and spleen of Spalax and rat subjected to 6% O2 or normoxia. We identified differentially expressed genes and pathways common to multiple organs in Spalax and rat. Body-wide differences between Spalax and rat affected biological processes like cell death, defence against reactive oxygen species (ROS), DNA repair, energy metabolism, immune response and angiogenesis, which altogether might play a crucial role in Spalaxs adaptation to life under oxygen deprivation. In all organs, mRNA expression of genes associated with genome stability maintenance and DNA repair was elevated in Spalax compared to rat, accompanied by a lower gene expression of genes associated with aerobic energy metabolism and proinflammatory processes. These transcriptomic changes might be accountable for the extraordinary lifespan of Spalax and its cancer resistance. Our results reveal gene regulatory networks that become candidates for the investigation of the molecular bases that underlie the complex phenotype of Spalax.

Cells respond to stress by synthesising chaperone proteins that correct protein misfolding to maintain function. However, protein homeostasis is lost in ageing, leading to aggregates characteristic of protein-folding diseases. Whilst much is known about how these diseases progress, discovering what causes protein-folding to deteriorate could be key to their prevention. Here, we examined primary human mesenchymal stem cells (hMSCs), cultured to a point of replicative senescence and subjected to heat shock, as an in vitro model of the ageing stress response. We found through proteomic analysis that the maintenance of homeostasis deteriorated in senescent cells. Time-resolved analysis of factors regulating heat shock protein 70 kDa (HSPA1A) revealed a lack of capacities for protein turnover and translation to be key factors in limiting the stress response during senescence. A kinetic model predicted a consequence of these reduced capacities to be the accumulation of misfolded protein, a hypothesis supported by evidence of systematic changes to protein fold state. These results thus further our understanding of the underlying mechanistic links between ageing and loss of protein homeostasis.

Co-translational degradation via the ubiquitin-proteasome system mediates quality control of 15 - 25% of nascent proteins, a proportion that is known to increase dramatically as a result of proteotoxic stress. Whereas the ubiquitylation machinery involved has been characterized, mechanisms coordinating the proteasomal destruction of ribosome-attached nascent proteins remain poorly defined. In pursuit of such mechanisms, we discovered dual cooperation of the HSP70 family member HSPA1 with the 26S proteasome: First, in response to proteotoxic stress, HSPA1 promotes proteasome recruitment to translating 80S ribosomes in a manner independent of nascent chain ubiquitylation. Secondly, HSPA1, in association with its cognate nucleotide exchange factor HSPH1, maintains co-translationally ubiquitylated proteins in a soluble state required for efficient proteasomal degradation. Both mechanisms conspire to confer thermotolerance to cells and to promote the growth of esophageal cancer cells in vitro and in animals. Consistent with these observations, HSPH1 knockout impedes tumor growth in vitro and in animals and correlates with favorable prognosis in digestive tract cancers, thus nominating HSPH1 as a cancer drug target. HighlightsO_LIProteotoxic stress causes translational arrest, co-translational protein ubiquitylation, and proteasome recruitment to ribosomes C_LIO_LICo-translational proteasome recruitment is independent of nascent chain ubiquitylation but is augmented by HSPA1 C_LIO_LIHSPA1-HSPH1 disaggregase confers thermotolerance by maintaining the solubility and proteasomal clearance of ubiquitylated proteins C_LIO_LILow HSPH1 impedes co-translational thermotolerance and tumor growth and correlates with favorable prognosis in various cancers C_LI

Ability to adapt to temperature changes trough the Heat Shock Response (HSR) pathways is one of the most fundamental and clinically relevant cellular response systems. Here we report that Heat Shock (HS) induces a temporally-coordinated and stimulus-specific adaptation of the signalling and gene expression responses of the Nuclear Factor {kappa}B (NF-{kappa}B) transcription factor. We show that exposure of MCF7 breast adenocarcinoma cells to 43{degrees}C 1h HS inhibits the immediate signalling response to pro-inflammatory Interleukin 1{beta} (IL1{beta}) and Tumour Necrosis Factor (TNF) cytokines. Within 4h after HS treatment IL1{beta}-induced responses return to normal levels, but the recovery of the TNF-induced responses is delayed. Using siRNA knock-down of Heat Shock Factor 1 and mathematical modelling we show that the stimulus-specificity is conferred via the Inhibitory {kappa}B kinase signalosome, with HSR differentially controlling individual cytokine transduction pathways. Finally, using a novel mathematical model we predict and experimentally validate that the HSR cross-talk confers differential cytokine sensitivity of the NF-{kappa}B system to a range of physiological and clinically-relevant temperatures. This quantitative understanding of NF-{kappa}B and HSR cross-talk mechanisms is fundamentally important for the potential improvement of current hyperthermia protocols.

Chloroplasts are the main energy organelles in plants, primary through photosynthesis. Thereby, they are responsible for CO2 fixation and dioxygen production, which are essential for living species on Earth. To ensure these processes, numerous proteins encoded from the nuclear DNA need to be imported inside the chloroplast, and eventually to the thylakoids. Whereas the translocation systems from both chloroplastic and thylakoids membranes have been studied in recent years, the stromal route between these two membranes is largely unknown. Notably, the chloroplastic HSP90 (HSP90C) is likely to play an important role in this process, but its structure and molecular mechanisms remain to be unveiled. In this study, we used a combination of structural and biophysical approaches to elucidate the features of Arabidopsis thalianas HSP90C. Principally, we found that HSP90C has a remarkably high ATPase activity among the HSP90 family proteins. Further investigation allowed us to pinpoint atypical mechanisms responsible for this high activity. First, the N-terminal cap is involved in a disulfide bond that accelerates the ATPase activity of HSP90C. Second, its C-terminal domain features an extension that is mandatory for its dimerization. Third, our crystal structures reveal a wide opening of the HSP90Cs dimer with reduced intermonomeric interfaces. Lastly, we identified a helical switch which is required for HSP90Cs high activity. Three of these four features are due to sequence signatures of HSP90C, which we found to be shared by most of green plants representatives. Our study provides first insights of HSP90Cs non-canonical mechanisms, which will help in the understanding of processes related to protein import in the chloroplast.

Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain, and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 uM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection. Cellular protection was protective via the antioxidant proteins catalase and GGCS.

Cold environments pose substantial metabolic challenges to ectothermic organisms. In amphibians, such as the African clawed frog (Xenopus laevis), exposure to cold temperatures induces pronounced hyperglycemia; however, the molecular mechanisms underlying this response remain unclear. This study investigated the metabolic responses of the liver to cold exposure using transcriptome analysis. Adult frogs were subjected to a temperature of 5{degrees}C for five days, and their liver transcriptome was subsequently analyzed using RNA sequencing. Cold exposure significantly elevated blood glucose levels. Transcriptome analysis revealed extensive alterations in gene expression, including the upregulation of key gluconeogenesis-related genes. Notably, genes involved in FOXO1 signaling exhibited coordinated changes, with increased expression of foxo1 and its regulator prmt1 (arginine methyltransferase) and decreased expression of mdm2 (E3 ubiquitin ligase), suggesting that the phosphorylation of FOXO1 may be suppressed. Consistent with these findings, the expression of gluconeogenic genes (g6pc1 and pck1) was elevated, whereas the glycolytic gene gck was downregulated, indicating a shift towards glucose production. In addition to carbohydrate metabolism, genes involved in lipid and cholesterol metabolism, particularly fatty acid desaturases (scd and fads2), were also upregulated, suggesting that the remodeling of membrane lipid composition may occur under cold conditions. Furthermore, genes related to antioxidant and redox pathways, including those involved in the detoxification of reactive oxygen species and iron sequestration, were induced, indicating enhanced redox regulation. Collectively, these results demonstrate that cold exposure induces coordinated metabolic remodeling in the liver of X. laevis, characterized by enhanced gluconeogenesis, lipid remodeling, and robust redox regulation. SUMMARY STATEMENTCold exposure drives coordinated hepatic metabolic reprogramming in Xenopus laevis, elevating gluconeogenesis, modifying lipid composition, and strengthening antioxidant defenses through integrated transcriptional responses that support survival under a low-temperature environment.

Redox balance is central to aerobic metabolism, yet acute heat stress can destabilize this balance by increasing metabolic rates and shifting the balance of critical electron carriers such as NADH. In early Drosophila melanogaster embryos, maintaining redox balance is particularly critical as embryos undergo a developmental redox shift and rely on oxidative phosphorylation to power nuclear divisions. Here, we assayed six isofemale D. melanogaster lines from temperate (Vermont, USA; France; Japan) and tropical (St. Kitts; Ghana; India) climates to assess metabolic responses to heat in heat-sensitive versus heat-tolerant embryos. We used untargeted LC--MS to measure 33 metabolites and the major redox couples (NADH/NAD+, NADPH/NADP+, and GSH/GSSG) at 25{degrees}C and after a 32{degrees}C heat shock. In all embryos, heat shock induced shared shifts in metabolic profiles, with increases in nucleotide monophosphates (e.g., AMP, CMP, and GMP) and amino acids (e.g., alanine, glutamic acid, serine). In contrast, redox metabolites diverged by region: heat-sensitive temperate embryos shifted toward a more oxidized state (46.6% decrease in NADH/NAD+ ratio and 4-fold increase in oxidized glutathione), while heat-tolerant tropical embryos maintained glutathione balance and increased the NADH/NAD+ ratio by 52.9%, indicating a more reduced state. These patterns are consistent with higher NADH oxidation and greater oxidative stress (inferred from oxidized glutathione) in the temperate embryos, versus better maintenance of redox balance in tropical embryos. Together, our results suggest that maintaining redox balance is a key determinant of acute heat tolerance, and healthy development overall, during early embryogenesis.

Acquiring new cellular states entails metabolic reprogramming driven by changes in the expression of cytosolic and mitochondrial metabolic enzymes. Most mitochondrial proteins are synthesized in the cytosol and imported into the mitochondria in a linear form, after which they are folded by a network of mitochondrial chaperones and co-chaperones. Which mitochondrial protein is dependent upon which chaperone for its folding is largely unknown. HSPD1/HSPE1 (HSP60/HSP10) are evolutionarily conserved mammalian homologues of the bacterial proteins GroEL/GroES, forming a chamber-and-lid chaperonin to facilitate the folding of client proteins. We used gene knockdown and SILAC-based proteomics to identify HSPD1 client proteins. We found that HSPD1 supports the expression of Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2), a key essential protein in the mitochondrial one-carbon (1C) pathway, in cells and tumors, and directly folds MTHFD2, independently of its co-chaperone HSPE1. HSPD1 interacts with MTHFD2 in mitochondria, and MTHFD2 is degraded by LONP1 in HSPD1 knockdown cells. Consequently, we observed reduced nucleotide and S-adenosylmethionine (SAM) levels in HSPD1 knockdown and found minimal overlap in the transcriptional and metabolic cellular responses to HSPD1 vs. HSPE1 depletion. In C. elegans, knockout of HSP60 triggers the mitochondrial stress response in the gut, while HSP10 knockout triggers the mitochondrial stress response in muscle tissue. Our data support that HSPD1 is an MTHFD2 chaperone and that, in addition to working together, HSPD1 and HSPE1 have distinct biological functions.

Plants employ diverse strategies to cope with different types of heat stress. The response to short-term acute heat stress differs significantly from that to moderate heat stress followed by severe stress events. After experiencing moderate heat stress, plants exhibit a more robust response to subsequent severe stress, a phenomenon known as thermopriming or acquired thermotolerance. Thermopriming creates a memory by maintaining the heat stress (HS) memory-related genes in an alert state. In this work, we investigated the role of Arabidopsis Universal Stress Protein 1 (USP1) in plant heat stress responses. CRISPR-Cas9 generated knockout usp1 mutant lines showed no morphological changes during development and normal growth conditions. However, usp1 mutant plants showed enhanced levels of apoplast hydrogen peroxide and superoxide reactive oxygen species accumulation upon heat stress. Transcriptome analyses revealed that genes related to protein folding, electron transport, and oxidative phosphorylation are strongly upregulated in usp1 mutant plants. USP1 is essential for acquired thermotolerance, as usp1 mutants are compromised in heat stress memory but show normal responses to acute heat stress similar to hsfa2 mutants. Biochemical assays showed that USP1 functions as a molecular chaperone, protecting the transcription factor HSFA2 from heat-induced denaturation. Moreover, usp1 mutant plants show decreased transcript levels of heat stress response genes and reduced H3K4me3 enrichment at memory gene loci. These data show that USP1 plays an important role as a chaperone of HSFA2 in mediating plant heat stress memory.

The exposure to environmental stressors early in life can shape organisms to express more tolerant phenotypes to the same conditions during adulthood, a process called developmental plasticity. However, this acquired acclimation ability might depend on the intensity of the stimulus perceived later in life. Here, we took advantage of a purple sea urchin Paracentrotus lividus population developed at abnormally high sea temperature in the proximity of a power plant to test the limits of their developmentally acquired plasticity to increased water temperature. We simulated two marine heatwaves, a category I (moderate) and IV (extreme), and exposed the power plant population as well as a naive population developed in natural sea conditions. We measured their respiration rate and molecular responses to these two heatwaves. Regardless of the population of origin, sea urchins exposed to heatwaves showed higher oxygen consumption, indicating an increase in metabolic rates. At the molecular level, the biggest difference between the two populations was found following the moderate heatwave. Compared to the developmentally acclimated sea urchins, the naive population expressed genes coding for proteins with stress response, chromatin remodeling and RNA splicing functions, while suppressing immune response, revealing that developmental exposures can aid in priming the responses of adults to moderate temperature increases. However, a stronger heatwave leveled the differences between the two populations, with sea urchins from both locations expressing genes involved in proteostasis and detoxification. Nevertheless, regardless of the simulated marine heatwave intensity, sea urchins from the naive population always showed enrichment of the spliceosome pathway compared to power plant urchins, which activated immune response genes instead, reflecting fundamentally different thermal stress-coping strategies shaped by their developmental environments. Overall, our results demonstrate the critical yet context-dependent role of developmental plasticity in shaping the resilience of marine ectotherms to climate change.

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate-early gene (IEG) induced by stress and synaptic activity, characterized by transient expression and rapid degradation. However, the mechanisms governing its degradation remain unclear. In this study, we identify a novel degradation pathway for Arc/Arg3.1, driven by its structural features. We demonstrate that the proteasomal degradation of Arc/Arg3.1 is modulated by the Hsp70-CHIP complex, with ubiquitination being impaired in HSF1 knockout cells. The formation of a Cys34-Cys159 disulfide bond crosslinks Arc/Arg3.1 into high-molecular-weight oligomers, altering its ubiquitination pattern and degradation kinetics compared to the C159A mutant. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed that wild-type (WT) Arc/Arg3.1 adopts a more compact structure than the C159A mutant. Notably, the C159A mutant fails to interact with HSF1, resulting in Hsp70 induction upon heat shock. Our findings propose a feedback loop in which disulfide crosslinking of Arc/Arg3.1 induces rapid degradation through Hsp70-mediated ubiquitination, which in turn modulates the heat shock response by inhibiting HSF1 function.