Genome-wide identification, classification and expression analysis of the heat shock transcription factor family in Garlic (Allium sativum L.).

BACKGROUND: The heat shock transcription factor (HSF) plays a crucial role in the regulatory network by coordinating responses to heat stress as well as other stress signaling pathways. Despite extensive studies on HSF functions in various plant species, our understanding of this gene family in garlic, an important crop with nutritional and medicinal value, remains limited. In this study, we conducted a comprehensive investigation of the entire garlic genome to elucidate the characteristics of the AsHSF gene family. RESULTS: In this study, we identified a total of 17 AsHSF transcription factors. Phylogenetic analysis classified these transcription factors into three subfamilies: Class A (9 members), Class B (6 members), and Class C (2 members). Each subfamily was characterized by shared gene structures and conserved motifs. The evolutionary features of the AsHSF genes were investigated through a comprehensive analysis of chromosome location, conserved protein motifs, and gene duplication events. These findings suggested that the evolution of AsHSF genes is likely driven by both tandem and segmental duplication events. Moreover, the nucleotide diversity of the AsHSF genes decreased by only 0.0002% from wild garlic to local garlic, indicating a slight genetic bottleneck experienced by this gene family during domestication. Furthermore, the analysis of cis-acting elements in the promoters of AsHSF genes indicated their crucial roles in plant growth, development, and stress responses. qRT-PCR analysis, co-expression analysis, and protein interaction prediction collectively highlighted the significance of Asa6G04911. Subsequent experimental investigations using yeast two-hybridization and yeast induction experiments confirmed its interaction with HSP70/90, reinforcing its significance in heat stress. CONCLUSIONS: This study is the first to unravel and analyze the AsHSF genes in garlic, thereby opening up new avenues for understanding their functions. The insights gained from this research provide a valuable resource for future investigations, particularly in the functional analysis of AsHSF genes.

Metformin induction of heat shock factor 1 activation and the mitochondrial unfolded protein response alleviate cardiac remodeling in spontaneously hypertensive rats.

Heart failure and cardiac remodeling are both characterized by mitochondrial dysfunction. Healthy mitochondria are required for adequate contractile activity and appropriate regulation of cell survival. In the mammalian heart, enhancement of the mitochondrial unfolded protein response (UPRmt) is cardioprotective under pressure overload conditions. We explored the UPRmt and the underlying regulatory mechanism in terms of hypertension-induced cardiac remodeling and the cardioprotective effect of metformin. Male spontaneously hypertensive rats and angiotensin II-treated neonatal rat cardiomyocytes were used to induce cardiac hypertrophy. The results showed that hypertension induced the formation of aberrant mitochondria, characterized by a reduced mtDNA/nDNA ratio and swelling, as well as lower levels of mitochondrial complexes I to V and inhibition of the expression of one protein subunit of each of complexes I to IV. Such changes eventually enlarged cardiomyocytes and increased cardiac fibrosis. Metformin treatment increased the mtDNA/nDNA ratio and regulated the UPRmt, as indicated by increased expression of activating transcription factor 5, Lon protease 1, and heat shock protein 60, and decreased expression of C/EBP homologous protein. Thus, metformin improved mitochondrial ultrastructure and function in spontaneously hypertensive rats. In vitro analyses revealed that metformin reduced the high levels of angiotensin II-induced mitochondrial reactive oxygen species in such animals and stimulated nuclear translocation of heat shock factor 1 (HSF1). Moreover, HSF1 small-interfering RNA reduced the metformin-mediated improvements in mitochondrial morphology and the UPRmt by suppressing hypertrophic signals and cardiomyocyte apoptosis. These results suggest that HSF1/UPRmt signaling contributes to the beneficial effects of metformin. Metformin-mediated targeting of mitochondrial protein homeostasis and modulation of HSF1 levels have potential therapeutic implications in terms of cardiac remodeling.

Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

Heat shock factor 1 inhibition enhances the effects of modulated electro hyperthermia in a triple negative breast cancer mouse model.

Female breast cancer is the most diagnosed cancer worldwide. Triple negative breast cancer (TNBC) is the most aggressive type and there is no existing endocrine or targeted therapy. Modulated electro-hyperthermia (mEHT) is a non-invasive complementary cancer therapy using an electromagnetic field generated by amplitude modulated 13.56 MHz frequency that induces tumor cell destruction. However, we have demonstrated a strong induction of the heat shock response (HSR) by mEHT, which can result in thermotolerance. We hypothesized that inhibition of the heat shock factor 1 (HSF1) can synergize with mEHT and enhance tumor cell-killing. Thus, we either knocked down the HSF1 gene with a CRISPR/Cas9 lentiviral construct or inhibited HSF1 with a specific small molecule inhibitor: KRIBB11 in vivo. Wild type or HSF1-knockdown 4T1 TNBC cells were inoculated into the mammary gland's fat pad of BALB/c mice. Four mEHT treatments were performed every second day and the tumor growth was followed by ultrasound and caliper. KRIBB11 was administrated intraperitoneally at 50 mg/kg daily for 8 days. HSF1 and Hsp70 expression were assessed. HSF1 knockdown sensitized transduced cancer cells to mEHT and reduced tumor growth. HSF1 mRNA expression was significantly reduced in the KO group when compared to the empty vector group, and consequently mEHT-induced Hsp70 mRNA upregulation diminished in the KO group. Immunohistochemistry (IHC) confirmed the inhibition of Hsp70 upregulation in mEHT HSF1-KO group. Demonstrating the translational potential of HSF1 inhibition, combined therapy of mEHT with KRIBB11 significantly reduced tumor mass compared to either monotherapy. Inhibition of Hsp70 upregulation by mEHT was also supported by qPCR and IHC. In conclusion, we suggest that mEHT-therapy combined with HSF1 inhibition can be a possible new strategy of TNBC treatment with great translational potential.

Atypical heat shock transcription factor HSF5 is critical for male meiotic prophase under non-stress conditions.

Meiotic prophase progression is differently regulated in males and females. In males, pachytene transition during meiotic prophase is accompanied by robust alteration in gene expression. However, how gene expression is regulated differently to ensure meiotic prophase completion in males remains elusive. Herein, we identify HSF5 as a male germ cell-specific heat shock transcription factor (HSF) for meiotic prophase progression. Genetic analyzes and single-cell RNA-sequencing demonstrate that HSF5 is essential for progression beyond the pachytene stage under non-stress conditions rather than heat stress. Chromatin binding analysis in vivo and DNA-binding assays in vitro suggest that HSF5 binds to promoters in a subset of genes associated with chromatin organization. HSF5 recognizes a DNA motif different from typical heat shock elements recognized by other canonical HSFs. This study suggests that HSF5 is an atypical HSF that is required for the gene expression program for pachytene transition during meiotic prophase in males.

A wheat heat shock transcription factor gene, TaHsf-7A, regulates seed dormancy and germination.

Heat shock transcription factors (Hsfs) play multifaceted roles in plant growth, development, and responses to environmental factors. However, their involvement in seed dormancy and germination processes has remained elusive. In this study, we identified a wheat class B Hsf gene, TaHsf-7A, with higher expression in strong-dormancy varieties compared to weak-dormancy varieties during seed imbibition. Specifically, TaHsf-7A expression increased during seed dormancy establishment and subsequently declined during dormancy release. Through the identification of a 1-bp insertion (ins)/deletion (del) variation in the coding region of TaHsf-7A among wheat varieties with different dormancy levels, we developed a CAPS marker, Hsf-7A-1319, resulting in two allelic variations: Hsf-7A-1319-ins and Hsf-7A-1319-del. Notably, the allele Hsf-7A-1319-ins correlated with a reduced seed germination rate and elevated dormancy levels, while Hsf-7A-1319-del exhibited the opposite trend across 175 wheat varieties. The association of TaHsf-7A allelic status with seed dormancy and germination levels was confirmed in various genetically modified species, including Arabidopsis, rice, and wheat. Results from the dual luciferase assay demonstrated notable variations in transcriptional activity among transformants harboring distinct TaHsf-7A alleles. Furthermore, the levels of abscisic acid (ABA) and gibberellin (GA), along with the expression levels of ABA and GA biosynthesis genes, showed significant differences between transgenic rice lines carrying different alleles of TaHsf-7A. These findings represent a significant step towards a comprehensive understanding of TaHsf-7A's involvement in the dormancy and germination processes of wheat seeds.

Early signaling enhance heat tolerance in Arabidopsis through modulating jasmonic acid synthesis mediated by HSFA2.

Given the detrimental impact of global warming on crop production, it is particularly important to understand how plants respond and adapt to higher temperatures. Using the non-invasive micro-test technique and laser confocal microscopy, we found that the cascade process of early signals (K+, H2O2, H+, and Ca2+) ultimately resulted in an increase in the cytoplasmic Ca2+ concentration when Arabidopsis was exposed to heat stress. Quantitative real-time PCR demonstrated that heat stress significantly up-regulated the expression of CAM1, CAM3 and HSFA2; however, after CAM1 and CAM3 mutation, the upregulation of HSFA2 was reduced. In addition, heat stress affected the expression of LOX3 and OPR3, which was not observed when HSFA2 was mutated. Luciferase reporter gene expression assay and electrophoretic mobility shift assay showed that HSFA2 regulated the expression of both genes. Determination of jasmonic acid (JA) content showed that JA synthesis was promoted by heat stress, but was damaged when HSFA2 and OPR3 were mutated. Finally, physiological experiments showed that JA reduced the relative electrical conductivity of leaves, enhanced chlorophyll content and relative water content, and improved the survival rate of Arabidopsis under heat stress. Together, our results reveal a new pathway for Arabidopsis to sense and transmit heat signals; HSFA2 is involved in the JA synthesis, which can act as a defensive compound improving Arabidopsis heat tolerance.

Distinct induction pathways of heat shock protein 27 in human keratinocytes: Heat stimulation or capsaicin through phosphorylation of heat shock factor 1 at serine 326 and/or suppression of ΔNp63.

Epidermal keratinocytes, forming the outermost layer of the human body, serve as a crucial barrier against diverse external stressors such as ultraviolet radiation. Proper keratinocyte differentiation and effective responses to external stimuli are pivotal for maintaining barrier integrity. Heat is one such stimulus that triggers the synthesis of heat shock proteins (HSPs) when cells are exposed to temperatures above 42 °C. Additionally, activation of the transient receptor potential cation channel subfamily V member 1 (TRPV1) occurs at 42 °C. Here, we explore the interplay between TRPV1 signaling and HSP induction in human keratinocytes. Both heat and capsaicin, a TRPV1 agonist, induce expression of HSP27, HSP70, and HSP90 in keratinocytes. Interestingly, pharmacological inhibition of TRPV1 attenuates heat-induced HSP27 expression, but not that of HSP70 or HSP90. Furthermore, both heat and capsaicin stimulation result in distinct phosphorylation patterns of heat shock factor 1 (HSF1), with phosphorylation at serine 326 being a common feature. Notably, genetic manipulation to mimic dephosphorylation of HSF1 at serine 326 reduces HSP27 levels. Additionally, ΔNp63, a key regulator of epidermal differentiation, negatively modulates HSP27 expression independently of HSF1 phosphorylation status. While heat stimulation has no effect on ΔNp63 expression, capsaicin reduces its levels. The precise role of TRPV1 signaling in keratinocytes warrants further investigation for a comprehensive understanding of its impact on barrier function.

pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Comprehensive analysis of human tissues reveals unique expression and localization patterns of HSF1 and HSF2.

Heat shock factors (HSFs) are the main transcriptional regulators of the evolutionarily conserved heat shock response. Beyond cell stress, several studies have demonstrated that HSFs also contribute to a vast variety of human pathologies, ranging from metabolic diseases to cancer and neurodegeneration. Despite their evident role in mitigating cellular perturbations, the functions of HSF1 and HSF2 in physiological proteostasis have remained inconclusive. Here, we analyzed a comprehensive selection of paraffin-embedded human tissue samples with immunohistochemistry. We demonstrate that both HSF1 and HSF2 display distinct expression and subcellular localization patterns in benign tissues. HSF1 localizes to the nucleus in all epithelial cell types, whereas nuclear expression of HSF2 was limited to only a few cell types, especially the spermatogonia and the urothelial umbrella cells. We observed a consistent and robust cytoplasmic expression of HSF2 across all studied smooth muscle and endothelial cells, including the smooth muscle cells surrounding the vasculature and the high endothelial venules in lymph nodes. Outstandingly, HSF2 localized specifically at cell-cell adhesion sites in a broad selection of tissue types, such as the cardiac muscle, liver, and epididymis. To the best of our knowledge, this is the first study to systematically describe the expression and localization patterns of HSF1 and HSF2 in benign human tissues. Thus, our work expands the biological landscape of these factors and creates the foundation for the identification of specific roles of HSF1 and HSF2 in normal physiological processes.

Cellular HSF1 expression is induced during HIV-1 infection by activation of its promoter mediated through the cooperative interaction of HSF1 and viral Nef protein.

The Human Immunodeficiency Virus-1 (HIV-1) tends to activate cellular promoters driving expression of pro-viral genes by complex host-virus interactions for productive infection. We have previously demonstrated that expression of such a positive host factor HSF1 (heat shock factor 1) is elevated during HIV-1 infection; however, the mechanism remains to be elucidated. In the present study, we therefore examined whether HSF1 promoter is induced during HIV-1 infection leading to up-regulation of hsf1 gene expression. We mapped the putative transcription start site (TSS) predicted by Eukaryotic promoter database and deletion constructs of the predicted promoter region were tested through luciferase assay to identify the active promoter. The 347 bp upstream to 153 bp downstream region around the putative TSS displayed the highest activity and both Sp1 (stimulating protein 1) and HSF1 itself were identified to be important for its basal activation. Activity of HSF1 promoter was further stimulated during HIV-1 infection in CD4+ T cells, where interestingly the HSF1-site itself seems to play a major role. In addition, HIV-1 protein Nef (negative factor) was also observed to be responsible for the virus-mediated induction of hsf1 gene expression. Chromatin-immunoprecipitation assays further demonstrate that Nef and HSF1 are co-recruited to the HSF1-binding site and cooperatively act on this promoter. The interplay between host HSF1 and viral Nef on HSF1 promoter eventually leads to increase in HSF1 expression during HIV-1 infection. Understanding the mechanism of HSF1 up-regulation during HIV-1 infection might contribute to future antiviral strategies as HSF1 is a positive regulator of virus replication.

Magnolol extends lifespan and improves age-related neurodegeneration in Caenorhabditis elegans via increase of stress resistance.

Magnolol is a naturally occurring polyphenolic compound in many edible plants, which has various biological effects including anti-aging and alleviating neurodegenerative diseases. However, the underlying mechanism on longevity is uncertain. In this study, we investigated the effect of magnolol on the lifespan of Caenorhabditis elegans and explored the mechanism. The results showed that magnolol treatment significantly extended the  lifespan of nematode and alleviated senescence-related decline in the nematode model. Meanwhile, magnolol enhanced stress resistance to heat shock, hydrogen peroxide (H2O2), mercuric potassium chloride (MeHgCl) and paraquat (PQ) in nematode. In addition, magnolol reduced reactive oxygen species and malondialdehyde (MDA) levels, and increased superoxide dismutase and catalase (CAT) activities in nematodes. Magnolol also up-regulated gene expression of sod-3, hsp16.2, ctl-3, daf-16, skn-1, hsf-1, sir2.1, etc., down-regulated gene expression of daf-2, and promoted intranuclear translocation of daf-16 in nematodes. The lifespan-extending effect of magnolol were reversed in insulin/IGF signaling (IIS) pathway-related mutant lines, including daf-2, age-1, daf-16, skn-1, hsf-1 and sir-2.1, suggesting that IIS signaling is involved in the modulation of longevity by magnolol. Furthermore, magnolol improved the age-related neurodegeneration in PD and AD C. elegans models. These results indicate that magnolol may enhance lifespan and health span through IIS and sir-2.1 pathways. Thus, the current findings implicate magnolol as a potential candidate to ameliorate the symptoms of aging.

Cloning and characterization of heat shock transcription factor 1 and its functional role for Hsp70 production in the sea slug Onchidium reevesii.

To investigate the regulatory role of heat shock transcription factor 1 of sea slug Onchidium reevesii (OrHSF1) on Hsp70 expression in the sea slug under stress , the OrHSF1 gene was cloned and bioinformatics analysis was performed, then the gene and protein expressions by RNA interference (RNAi) mediated knockdown of OrHSF1 expression were measured to clarify the regulatory relationship between OrHSF1 and Hsp70 under low-frequency noise (LFN) stress. Our study was the first to clone a 1572 bp sequence of the OrHSF1 gene, with the sequence coding for amino acids (CDS) being 729 bp, encoding 243 amino acids. O. reevesii shared a close evolutionary relationship with mollusks such as the Aplysia californica. OrHSF1 gene is widely expressed in different tissues of sea slugs, with the highest expression in the intestine and the lowest in the reproductive glands. Furthermore, we used RNA interference (RNAi) as a tool to silence the OrHSF1 gene in the central nervous system (CNS) and the results indicated that gene silencing was occurring systematically in the CNS and the suppression of OrHSF1 expression by RNAi-mediated gene silencing altered the expression of Hsp70; besides, the expression trends of OrHSF1 gene and Hsp70 were consistent in the 3 and 5-day RNAi experiment. Moreover, in sea slugs injected with siHSF1 and exposed to LFN, the mRNA expression and protein expression of Hsp70 in the CNS were significantly decreased compared to the low-frequency noise group (P < 0.05). This study demonstrated that OrHSF1 regulates Hsp70 expression in marine mollusks under low-frequency noise, and HSF1-Hsp70 axis plays a key role in stress response.

Deletion of the transcription factors Hsf1, Msn2 and Msn4 in yeast uncovers transcriptional reprogramming in response to proteotoxic stress.

The response to proteotoxic stresses such as heat shock allows organisms to maintain protein homeostasis under changing environmental conditions. We asked what happens if an organism can no longer react to cytosolic proteotoxic stress. To test this, we deleted or depleted, either individually or in combination, the stress-responsive transcription factors Msn2, Msn4, and Hsf1 in Saccharomyces cerevisiae. Our study reveals a combination of survival strategies, which together protect essential proteins. Msn2 and 4 broadly reprogram transcription, triggering the response to oxidative stress, as well as biosynthesis of the protective sugar trehalose and glycolytic enzymes, while Hsf1 mainly induces the synthesis of molecular chaperones and reverses the transcriptional response upon prolonged mild heat stress (adaptation).

Dendrobium Nobile Alcohol Extract Extends the Lifespan of Caenorhabditis elegans via hsf-1 and daf-16.

Dendrobium nobile is a traditional Chinese herb with anti-inflammatory, antioxidant, and neuroprotective properties. However, its antiaging effects are unclear. Herein, we studied the aging-related functions and the mechanism of action of the alcohol extract of Dendrobium nobile (DnAE) in the model organism Caenorhabditis elegans. The results indicated that 1 mg/mL DnAE slowed lipofuscin accumulation, decreased the levels of reactive oxygen species, elevated superoxide dismutase activity, enhanced oxidative and heat stress resistance, extended the lifespan of nematodes, protected their dopamine neurons from 6-hydroxydopamine-induced neurodegeneration, and reduced Aß-induced neurotoxicity. DnAE upregulated the mRNA expression of the transcription factors DAF-16 and HSF-1, promoted the nuclear localization of DAF-16, and enhanced the fluorescence intensity of HSP-16.2. However, it had no effect on the lifespan of DAF-16 mutants. Thus, DnAE can significantly extend lifespan, enhance heat stress tolerance, and delay age-related diseases through a DAF-16-dependent pathway.

miR-330-5p Suppress Cell Growth and Invasion via Disrupting HSF4-mediated MACC1/STAT3 Pathway in Colorectal Cancer.

BACKGROUND: Recently, miRNAs are demonstrated to restrain mRNA translation through novel pattern with bind complementary sites in the coding sequence (CDS). Heat Shock Transcription Factor 4 (HSF4) has been newly described as a tumor-associated transcription factor. Therefore, the present study intends to explore miRNAs that bind CDS region of HSF4, and identify the function of their interactions in the malignant biological behavior of colorectal cancer (CRC). METHODS: Prognostic value of HSF4 and correlation between HSF4 and MACC1 expression were estimated via bioinformatics with the Cancer Genome Atlas (TCGA) data. HSF4 and downstream MACC1/STAT3 signaling cascade was characterized by immunoblotting. To characterize the effects of miR-330-5p and HSF4 on the malignant phenotype of CRC cells by functional experiments. The binding activity of miR-330-5p to coding sequence (CDS) of HSF4 was identified using DIANA-microT-CDS algorithm and dual-luciferase reporter assay. RESULTS: HSF4 was aberrantly overexpressed and associated with poor outcomes of CRC patients. Overexpression of HSF4 was correlated with Tumor Node Metastasis stage, and positively regulated malignant behaviors such as growth, migration, invasion of CRC cells. Moreover, miR-330-5p suppressed CRC cell growth, colony formation, migration and invasive. Interestingly, miR-330-5p recognized complementary sites within the HSF4 CDS region to reduce HSF4 expression. In rescue experiments, restoration of HSF4 expression functionally alleviated miR-330-5p-induced inhibition of cell growth, colon formation, invasion, and wound healing of CRC cells. HSF4 was associated positively with the well-known oncogenic factor MACC1 in TCGA cohort CRC samples, and knockdown of HSF4 resulted in downregulation of MACC1. In mechanism, MACC1 was suppressed upon miR-330-5p-induced downregulation of HSF4, leading to inactivation of phosphorylation of downstream STAT3. CONCLUSION: miR-330-5p suppresses tumors by directly inhibiting HSF4 to negatively modify activity of MACC1/STAT3 pathway.

Transcription factor CaHDZ15 promotes pepper basal thermotolerance by activating HEAT SHOCK FACTORA6a.

High temperature stress (HTS) is a serious threat to plant growth and development and to crop production in the context of global warming, and plant response to HTS is largely regulated at the transcriptional level by the actions of various transcription factors (TFs). However, whether and how homeodomain-leucine zipper (HD-Zip) TFs are involved in thermotolerance are unclear. Herein, we functionally characterized a pepper (Capsicum annuum) HD-Zip I TF CaHDZ15. CaHDZ15 expression was upregulated by HTS and abscisic acid in basal thermotolerance via loss- and gain-of-function assays by virus-induced gene silencing in pepper and overexpression in Nicotiana benthamiana plants. CaHDZ15 acted positively in pepper basal thermotolerance by directly targeting and activating HEAT SHOCK FACTORA6a (HSFA6a), which further activated CaHSFA2. In addition, CaHDZ15 interacted with HEAT SHOCK PROTEIN 70-2 (CaHsp70-2) and glyceraldehyde-3-phosphate dehydrogenase1 (CaGAPC1), both of which positively affected pepper thermotolerance. CaHsp70-2 and CaGAPC1 promoted CaHDZ15 binding to the promoter of CaHSFA6a, thus enhancing its transcription. Furthermore, CaHDZ15 and CaGAPC1 were protected from 26S proteasome-mediated degradation by CaHsp70-2 via physical interaction. These results collectively indicate that CaHDZ15, modulated by the interacting partners CaGAPC1 and CaHsp70-2, promotes basal thermotolerance by directly activating the transcript of CaHSFA6a. Thus, a molecular linkage is established among CaHsp70-2, CaGAPC1, and CaHDZ15 to transcriptionally modulate CaHSFA6a in pepper thermotolerance.

Upregulation of Wheat Heat Shock Transcription Factor TaHsfC3-4 by ABA Contributes to Drought Tolerance.

Drought stress can seriously affect the yield and quality of wheat (Triticum aestivum). So far, although few wheat heat shock transcription factors (Hsfs) have been found to be involved in the stress response, the biological functions of them, especially the members of the HsfC (heat shock transcription factor C) subclass, remain largely unknown. Here, we identified a class C encoding gene, TaHsfC3-4, based on our previous omics data and analyzed its biological function in transgenic plants. TaHsfC3-4 encodes a protein containing 274 amino acids and shows the basic characteristics of the HsfC class. Gene expression profiles revealed that TaHsfC3-4 was constitutively expressed in many tissues of wheat and was induced during seed maturation. TaHsfC3-4 could be upregulated by PEG and abscisic acid (ABA), suggesting that this Hsf may be involved in the regulation pathway depending on ABA in drought resistance. Further results represented that TaHsfC3-4 was localized in the nucleus but had no transcriptional activation activity. Notably, overexpression of TaHsfC3-4 in Arabidopsis thaliana pyr1pyl1pyl2pyl4 (pyr1pyl124) quadruple mutant plants complemented the ABA-hyposensitive phenotypes of the quadruple mutant including cotyledon greening, root elongation, seedling growth, and increased tolerance to drought, indicating positive roles of TaHsfC3-4 in the ABA signaling pathway and drought tolerance. Furthermore, we identified TaHsfA2-11 as a TaHsfC3-4-interacting protein by yeast two-hybrid (Y2H) screening. The experimental data show that TaHsfC3-4 can indeed interact with TaHsfA2-11 in vitro and in vivo. Moreover, transgenic Arabidopsis TaHsfA2-11 overexpression lines exhibited enhanced drought tolerance, too. In summary, our study confirmed the role of TaHsfC3-4 in response to drought stress and provided a target locus for marker-assisted selection breeding to improve drought tolerance in wheat.

Novel mechanism of drug resistance triggered by tumor-associated macrophages through Heat Shock Factor-1 activation.

Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism.

Heat shock response during the resolution of inflammation and its progressive suppression in chronic-degenerative inflammatory diseases.

The heat shock response (HSR) is a crucial biochemical pathway that orchestrates the resolution of inflammation, primarily under proteotoxic stress conditions. This process hinges on the upregulation of heat shock proteins (HSPs) and other chaperones, notably the 70 kDa family of heat shock proteins, under the command of the heat shock transcription factor-1. However, in the context of chronic degenerative disorders characterized by persistent low-grade inflammation (such as insulin resistance, obesity, type 2 diabetes, nonalcoholic fatty liver disease, and cardiovascular diseases) a gradual suppression of the HSR does occur. This work delves into the mechanisms behind this phenomenon. It explores how the Western diet and sedentary lifestyle, culminating in the endoplasmic reticulum stress within adipose tissue cells, trigger a cascade of events. This cascade includes the unfolded protein response and activation of the NOD-like receptor pyrin domain-containing protein-3 inflammasome, leading to the emergence of the senescence-associated secretory phenotype and the propagation of inflammation throughout the body. Notably, the activation of the NOD-like receptor pyrin domain-containing protein-3 inflammasome not only fuels inflammation but also sabotages the HSR by degrading human antigen R, a crucial mRNA-binding protein responsible for maintaining heat shock transcription factor-1 mRNA expression and stability on heat shock gene promoters. This paper underscores the imperative need to comprehend how chronic inflammation stifles the HSR and the clinical significance of evaluating the HSR using cost-effective and accessible tools. Such understanding is pivotal in the development of innovative strategies aimed at the prevention and treatment of these chronic inflammatory ailments, which continue to take a heavy toll on global health and well-being.