Heat stress due to global warming adversely affects plant physiology and metabolism, significantly reducing agricultural productivity. Plants have evolved various adaptive mechanisms to cope with such stresses, involving a range of heat stress-responsive proteins. This study investigates the molecular functions and structural changes of OsTDX (Oryza sativa TPR repeat-containing thioredoxin) in rice under heat stress, focusing on its roles as a disulfide reductase and molecular chaperone. OsTDX, sharing a 52 % overall amino acid identity with AtTDX, predominantly forms high molecular weight (HMW) complexes under heat stress conditions. Functional assays revealed that OsTDX exhibited increased disulfide reductase activity in a dose-dependent manner and significantly enhanced holdase chaperone activity, particularly under specific heat stress conditions (60 °C). The structural shift from low molecular weight (LMW) to HMW forms was accompanied by increased hydrophobicity, as indicated by bis-ANS fluorescence intensity measurements. In conclusion, OsTDX exhibits dual functions as a disulfide reductase and a holdase chaperone, with its chaperone activity significantly enhanced under heat stress through structural changes to HMW complexes. These findings contribute to understand the molecular mechanisms of heat tolerance in rice and highlight the potential role of OsTDX in the development of heat-tolerant crops to address crop yield declines due to global warming.

Heat shock factor 1 (HSF1) responds to stress to mount the heat shock response (HSR), a conserved transcriptional program that allows cells to maintain proteostasis by upregulating heat shock proteins (HSPs). The homeostatic stress regulation of HSF1 plays a key role in human physiology and health but its mechanism has remained difficult to pinpoint. Recent work in the budding yeast model has implicated stress-inducible chaperones of the HSP70 family as direct negative regulators of HSF1 activity. Here, we have investigated the latency control and activation of human HSF1 by HSP70 and misfolded proteins. Purified oligomeric HSF1-HSP70 (HSPA1A) complexes exhibited basal DNA binding activity that was inhibited by increasing the levels of HSP70 and, importantly, misfolded proteins reverted the inhibitory effect. Using site-specific UV photo-crosslinking, we monitored HSP70-HSF1 complexes in HEK293T cells. While HSF1 was bound by the substrate binding domain of HSP70 in unstressed cells, activation of HSF1 by heat shock as well as by inducing the misfolding of newly synthesized proteins resulted in release of HSF1 from the chaperone. Taken our results together, we conclude that latent HSF1 populate dynamic complexes with HSP70, which are sensitive to increased levels of misfolded proteins that compete for binding to the HSP70 substrate binding domain. Thus, human HSF1 is activated by various stress conditions that all titrate available HSP70.

RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.

Intron retention (IR) constitutes a less explored form of alternative splicing, wherein introns are retained within mature mRNA transcripts. This investigation demonstrates that the cell division cycle (CDC)-like kinase 2 (CLK2) undergoes liquid-liquid phase separation (LLPS) within nuclear speckles in response to heat shock (HS). The formation of CLK2 condensates depends on the intrinsically disordered region (IDR) located within the N-terminal amino acids 1-148. Phosphorylation at residue T343 sustains CLK2 kinase activity and promotes overall autophosphorylation, which inhibits the LLPS activity of the IDR. These CLK2 condensates initiate the reorganization of nuclear speckles, transforming them into larger, rounded structures. Moreover, these condensates facilitate the recruitment of splicing factors into these compartments, restricting their access to mRNA for intron splicing and promoting the IR. The retained introns lead to the sequestration of transcripts within the nucleus. These findings extend to the realm of glioma stem cells (GSCs), where a physiological state mirroring HS stress inhibits T343 autophosphorylation, thereby inducing the formation of CLK2 condensates and subsequent IR. Notably, expressing the CLK2 condensates hampers the maintenance of GSCs. In conclusion, this research unveils a mechanism by which IR is propelled by CLK2 condensates, shedding light on its role in coping with cellular stress.

Cloning and expression of a gene in the desired host is required for optimum production in recombinant strains. The present research is the first attempt to optimize the physiological conditions for the transformation of Pseudomonas aeruginosa SDK-6 with pJN105. Different factors, such as inoculum size, incubation period, heat shock temperature, and heat shock time were optimized using one factor at a time (OFAT) followed by the selection of transformants using gentamicin resistance marker. The maximum number of transformants (2.002 ± 0.077 × 105 cfu/ µg of plasmid DNA) were reported with 0.5% (v/v) inoculum, an incubation period of 3 h, and heat shock treatment at 50 °C for 1 min. An overall 12-fold increase in transformation efficiency was observed. The presence of a 6055 bp band on agarose gel confirmed the transformation of Pseudomonas aeruginosa with the vector pJN105.

Grain filling is a critical process for improving crop production under adverse conditions caused by climate change. Here, using a quantitative method, we quantified post-anthesis source-sink relationships of a large data set to assess the contribution of remobilized pre-anthesis assimilates to grain growth for both biomass and nitrogen. The data set came from 13 years\' semi-controlled field experimentation, in which six bread wheat genotypes were grown at plot scale under contrasting temperature, water, and nitrogen regimes. On average, grain biomass was ~10% higher than post-anthesis aboveground biomass accumulation across regimes and genotypes. Overall, the estimated relative contribution (%) of remobilized assimilates to grain biomass became increasingly significant with increasing stress intensity, ranging from virtually nil to 100%. This percentage was altered more by water and nitrogen regimes than by temperature, indicating the greater impact of water or nitrogen regimes relative to high temperatures under our experimental conditions. Relationships between grain nitrogen demand and post-anthesis nitrogen uptake were generally insensitive to environmental conditions, as there was always significant remobilization of nitrogen from vegetative organs, which helped to stabilize the amount of grain nitrogen. Moreover, variations in the relative contribution of remobilized assimilates with environmental variables were genotype-dependent. Our analysis provides an overall picture of post-anthesis source-sink relationships and pre-anthesis assimilate contributions to grain filling across (non-)environmental factors, and highlights that designing wheat adaption to climate change should account for complex multi-factor interactions.

Nucleoli form interchromosomal contacts with genes controlling differentiation and carcinogenesis. DUX4 genes specify transcription factor possessing two homeodomains. Previously, using Circular Chromosome Conformation Capture (4С) approach on population of cells, it was demonstrated that DUX4 gene clusters form frequent contacts with nucleoli. It was found also that these contacts are almost completely abolished after heat shock treatment. 4C approach as all ligation-mediated methods is capable to detect rather close interactions between chromatin loops in nuclei. In order to independently confirm the formation and the frequency of the contacts in single cells we used FISH approach. Here, we show that DUX genes in single cells form stable contacts in all tested HEK293T cells. During heat shock, DUX4 genes reversibly move 1-3 µm away from the nuclei. We conclude that interchromosomal contacts formed by nucleoli are strong, dynamic, and reversible, providing both the initiation and maintenance of a differentiated state.

Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frame (sORF)-encoded polypeptides (SEPs) of fewer than fifty amino acids have been identified, and biological functions have been ascribed to an increasing number of SEPs from intergenic regions and small RNAs. However, despite numbering in the dozens in Escherichia coli, and hundreds to thousands in humans, same-strand nested sORFs that overlap protein coding genes in alternative reading frames remain understudied. In order to provide insight into this enigmatic class of unannotated genes, we characterized GndA, a 36-amino acid, heat shock-regulated SEP encoded within the +2 reading frame of the gnd gene in E. coli K-12 MG1655. We show that GndA pulls down components of respiratory complex I (RCI) and is required for proper localization of a RCI subunit during heat shock. At high temperature GndA deletion (ΔGndA) cells exhibit perturbations in cell growth, NADH+/NAD ratio, and expression of a number of genes including several associated with oxidative stress. These findings suggest that GndA may function in maintenance of homeostasis during heat shock. Characterization of GndA therefore supports the nascent but growing consensus that functional, overlapping genes occur in genomes from viruses to humans.

Protein SUMOylation is a prevalent stress-response posttranslational modification crucial for maintaining cellular homeostasis. Herein, we report that protein SUMOylation modulates cellular signaling mediated by cAMP, an ancient and universal stress-response second messenger. We identify K561 as a primary SUMOylation site in exchange protein directly activated by cAMP (EPAC1) via site-specific mapping of SUMOylation using mass spectrometry. Sequence and site-directed mutagenesis analyses reveal that a functional SUMO-interacting motif in EPAC1 is required for the binding of SUMO-conjugating enzyme UBC9, formation of EPAC1 nuclear condensate, and EPAC1 cellular SUMOylation. Heat shock-induced SUMO modification of EPAC1 promotes Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.

Heat shock proteins (HSPs), which function as chaperones, are activated in response to various environmental stressors. In addition to their role in diverse aspects of protein production, HSPs protect against harmful protein-related stressors. Calycosin exhibits numerous beneficial properties. This study aims to explore the protective effects of calycosin in the heart under heat shock and determine its underlying mechanism. H9c2 cells, western blot, TUNEL staining, flow cytometry, and immunofluorescence staining were used. The time-dependent effects of heat shock analyzed using western blot revealed increased HSP expression for up to 2[Formula: see text]h, followed by protein degradation after 4[Formula: see text]h. Hence, a heat shock damage duration of 4[Formula: see text]h was chosen for subsequent investigations. Calycosin administered post-heat shock demonstrated dose-dependent recovery of cell viability. Under heat shock conditions, calycosin prevented the apoptosis of H9c2 cells by upregulating HSPs, suppressing p-JNK, enhancing Bcl-2 activation, and inhibiting cleaved caspase 3. Calycosin also inhibited Fas/FasL expression and activated cell survival markers (p-PI3K, p-ERK, p-Akt), indicating their cytoprotective properties through PI3K/Akt activation and JNK inhibition. TUNEL staining and flow cytometry confirmed that calycosin reduced apoptosis. Moreover, calycosin reversed the inhibitory effects of quercetin on HSF1 and Hsp70 expression, illustrating its role in enhancing Hsp70 expression through HSF1 activation during heat shock. Immunofluorescence staining demonstrated HSF1 translocation to the nucleus following calycosin treatment, emphasizing its cytoprotective effects. In conclusion, calycosin exhibits pronounced protective effects against heat shock-induced damages by modulating HSP expression and regulating key signaling pathways to promote cell survival in H9c2 cells.