Stress-responsive Transcription Factor Research Articles

Antiviral therapy reduces hepatocellular carcinoma through suppressing hepatitis B virus replication may improve ER stress, mitochondrial and metabolic dysfunctions and decrease p62 in hybridized mice with single HBV transgene and miR-122.

Hepatitis B virus (HBV) hijacks autophagy for its replication. Nucleos(t)ide analogs (NUCs) treatment suppressed HBV replication and reduced hepatocellular carcinoma (HCC) incidence. However, the use of NUCs in chronic hepatitis B (CHB) patients with normal or minimally elevated serum alanine aminotransferase (ALT) levels is still debated. Animal models are crucial for studying the unanswered issue and evaluating new therapies. MicroRNA-122 (miR-122), which regulates fatty acid and cholesterol metabolism, is downregulated during hepatitis and HCC progression. The reciprocal inhibition of miR-122 with HBV highlights its role in HCC development as a tumor suppressor. By crossbreeding HBV-transgenic mice with miR-122 knockout mice, we generated a hybrid mouse model with a high incidence of HCC up to 89% and normal ALT levels before HCC. The model exhibited early-onset hepatic steatosis, progressive liver fibrosis, and impaired late-phase autophagy. Metabolomics and microarray analysis identified metabolic signatures, including dysregulation of lipid metabolism, inflammation, genomic instability, the Warburg effect, reduced TCA cycle flux, energy deficiency, and impaired free radical scavenging. Antiviral treatment reduced HCC incidence in hybrid mice by approximately 30-35% compared to untreated mice. This effect was linked to the activation of ER stress-responsive transcription factor ATF4, clearance of autophagosome cargo p62, and suppression of the CHOP-mediated apoptosis pathway. In summary, this study suggests that despite minimal ALT elevation, HBV replication can lead to liver injury. Endoplasmic reticulum stress, reduced miR-122 levels, mitochondrial and metabolic dysfunctions, blocking protective autophagy resulting in p62 accumulation, apoptosis, fibrosis, and HCC. Antiviral may improve the above-mentioned pathogenesis through HBV suppression.

SARS-CoV-2 variant-specific differences in inhibiting the effects of the PKR-activated integrated stress response

The integrated stress response (ISR) is a eukaryotic cell pathway that triggers translational arrest and the formation of stress granules (SGs) in response to various stress signals, including those caused by viral infections. The SARS-CoV-2 nucleocapsid protein has been shown to disrupt SGs, but SARS-CoV-2 interactions with other components of the pathway remains poorly characterized. Here, we show that SARS-CoV-2 infection triggers the ISR through activation of the eIF2α-kinase PKR while inhibiting a variety of downstream effects. In line with previous studies, SG formation was efficiently inhibited and the induced eIF2α phosphorylation only minimally contributed to the translational arrest observed in infected cells. Despite ISR activation and translational arrest, expression of the stress-responsive transcription factors ATF4 and CHOP was not induced in SARS-CoV-2 infected cells. Finally, we found variant-specific differences in the activation of the ISR between ancestral SARS-CoV-2 and the Delta and Omicron BA.1 variants in that Delta infection induced weaker PKR activation while Omicron infection induced higher levels of p-eIF2α, and greatly increased SG formation compared to the other variants. Our results suggest that different SARS-CoV-2 variants can affect normal cell functions differently, which can have an impact on pathogenesis and treatment strategies.

Genome-wide transcriptional profiling and physiological investigation elucidating the molecular mechanism of multiple abiotic stress response in Stevia rebaudiana Bertoni

Considering the major source of plant-derived low/non-calorie steviol glycosides (SGs), comprehensive physiological, biochemical, and deep transcriptional investigations were conducted to explicit deeper insight into multiple abiotic stress responses in Stevia rebaudiana. The physiological indicators including photosynthesis, chlorophyll, relative water content, shoot growth, electrolyte leakage, and SG biosynthesis were negatively impacted under drought (DS), followed by salinity (SS) and waterlogging (WS). Global transcriptional analysis revealed significant upregulated expression of the genes encoding for ROS detoxification (GST, SOD, APX, glutathione peroxidase), osmotic adjustment (alpha-trehalose-phosphate and S-adenosylmethionine decarboxylase), ion transporters (CAX, NHX, CNGS, VPPase, VATPase), water channel (PIP1, TIP) and abiotic stress-responsive candidate genes (LEA, HSPs, and Dehydrins) regulating abiotic stress response in S. rebaudiana. These inferences were complemented with predicted interactome network that revealed regulation of energy metabolism by key stress-responsive genes (GST, HKT1, MAPKs, P5CSs, PIP), transcription factors (HSFA2, DREB1A, DREB2A), and abiotic stress responsive pathways (ABA, ethylene, ion stress). This is the first detailed study to comprehend the molecular regulation of stress response and their interplay under DS, SS, and WS. The key genes and regulators can be functionally validated, and will facilitate targeted gene editing for genetic improvement of crop sustainability under changing environmental conditions in S. rebaudiana.

Molecular characterization of genes involved in tolerance of cadmium in Triticum aestivum (L.) under Cd stress

The NRAMPs (natural resistance-associated macrophage proteins) are major transporters for the absorption and transport of metals like Pb, Zn, Mn, Fe, and Cd in plants. While NRAMP gene family members have been extensively studied as metal transporters in model and other plants, little information has been reported on their role in Triticum aestivum, particularly in response to Cd stress. Current study reported 13 NRAMP candidates in the genome of T. aestivum. Phylogenetic analysis divided these into three clades. Motif and gene structure study showed that members in the same clades shared the same location and pattern, which further supported the phylogenetic analysis. The analysis of cis-acting elements in promoter sequences of NRAMP genes in wheat identified stress-responsive transcription factor binding sites. Multiple sequence alignment identified the conservation of important residues. Based on RNA-seq and qRT-PCR analysis, Cd stress-responsive variations of TaNRAMP gene expression were reported. This study provides comprehensive data to understand the TaNRAMP gene family, its features, and its expression, which will be a helpful framework for functional research.

PKA-Msn2/4-Shy1 cascade controls inhibition of proline utilization under wine fermentation models

Proline, which is a predominant amino acid in grape musts, is involved in the taste and flavor of foods and beverages. The yeast Saccharomyces cerevisiae poorly utilizes proline in wine-making processes, leading to a nitrogen deficiency during fermentation and proline accumulation in wine. Previous studies have shown that the protein kinase A (PKA) pathway is involved in inhibitory mechanisms of proline utilization. In this study, we screened the PKA pathway-related genes that regulate proline utilization. Using a yeast culture collection of disrupted strains associated with the downstream of the PKA cascade, we revealed that the stress-responsive transcription factor genes MSN2/4 regulate proline utilization. Moreover, we found that Msn2/4 up-regulate the SHY1 gene during the cell growth of the wine fermentation model, which may cause the inhibition of proline utilization. The SHY1-deleted strain of the commercial wine yeast clearly showed proline consumption and average ethanol production under the wine fermentation model. The present data indicate that the PKA-Msn2/4-Shy1 cascade controls the inhibition of proline utilization under wine-making processes. Our study could hold promise for the development of wine yeast strains that can efficiently reduce proline during wine fermentation.

TOR inactivation triggers heterochromatin formation in rDNA during glucose starvation

In response to environmental cues, such as nutrient starvation, living organisms modulate gene expression through mechanisms involving histone modifications. Specifically, nutrient depletion inactivates the TOR (target of rapamycin) pathway, leading to reduced expression of ribosomal genes. While these regulatory mechanisms are well elucidated in budding yeast Saccharomyces cerevisiae, their conservation across diverse organisms remains unclear. In this study, we demonstrate that fission yeast Schizosaccharomyces pombe cells repress ribosomal gene transcription through a different mechanism. TORC1, which accumulates in the rDNA region, dissociates upon starvation, resulting in enhanced methylation of H3K9 and heterochromatin formation, facilitated by dissociation of the stress-responsive transcription factor Atf1 and accumulation of the histone chaperone FACT. We propose that this mechanism might be adapted in mammals that possess Suv39H1 and HP1, which are absent in budding yeast.

HSF1 Inhibits Antitumor Immune Activity in Breast Cancer by Suppressing CCL5 to Block CD8+ T-cell Recruitment.

The stress-responsive transcription factor HSF1 reduces CD8+ T-cell infiltration in breast tumors to prevent immune-mediated killing, indicating that cellular stress responses affect tumor-immune interactions and that targeting HSF1 could improve immunotherapies.

The Gma-miR394a/GmFBX176 module is involved in regulating the soybean (Glycine max L.) response to drought stress

Drought seriously affects the yield and quality of soybean. Previous studies have shown that the gma-miR394a/GmFBX176 module regulates the response of Arabidopsis to drought stress. However, whether the gma-miR394a/GmFBX176 module is involved in the regulation of the soybean drought stress response remains unclear. Here, the function of the gma-miR394a/GmFBX176 module in the soybean drought stress response was evaluated. In soybean hairy roots, drought stress induced the transcription of gma-miR394a and inhibited the transcription of GmFBX176. GUS histochemical staining showed that transgenic GmFBX176p:GUS soybean hairy root staining was weak and that GUS transcript levels decreased under drought stress. A transient expression experiment in tobacco showed that gma-miR394a inhibited GmFBX176 transcription. Under drought stress, composite soybean plants overexpressing gma-miR394a showed increased drought resistance compared with control K599 composite soybean plants (K599); their survival rate and peroxidase activity were higher than those of K599, and their malondialdehyde content was lower. In contrast, composite soybean plants overexpressing GmFBX176m3 (gma-miR394a complement site mutation) presented lower drought resistance than K599 plants. Transcriptomic sequencing showed that the gma-miR394a/GmFBX176 module affected the transcript levels of stress response genes and transcription factors. These results indicate that the gma-miR394a/GmFBX176 module can be used to improve the drought resistance of soybean.

Network of GRAS transcription factors in plant development, fruit ripening and stress responses

Abstract The plant-specific family of GRAS transcription factors has been wide implicated in the regulation of transcriptional reprogramming associated with a diversity of biological functions ranging from plant development processes to stress responses. Functional analyses of GRAS transcription factors supported by in silico structural and comparative analyses are emerging and clarifying the regulatory networks associated with their biological roles. In this review, a detailed analysis of GRAS proteins' structure and biochemical features as revealed by recent discoveries indicated how these characteristics may impact subcellular location, molecular mechanisms, and function. Nomenclature issues associated with GRAS classification into different subfamilies in diverse plant species even in the presence of robust genomic resources are discussed, in particular how it affects assumptions of biological function. Insights into the mechanisms driving evolution of this gene family and how genetic and epigenetic regulation of GRAS contributes to subfunctionalization are provided. Finally, this review debates challenges and future perspectives on the application of this complex but promising gene family for crop improvement to cope with challenges of environmental transition.

A pervasive phosphorylation cascade modulation of plant transcription factors in response to abiotic stress.

Transcriptional regulation of stress-responsive genes is a crucial step in establishing the mechanisms behind plant abiotic stress tolerance. A sensitive method of regulating transcription factors activity, stability, protein interaction, and subcellular localization is through phosphorylation. This review highlights a widespread regulation mechanism that involves phosphorylation of plant TFs in response to abiotic stress. Abiotic stress is one of the main components limiting crop yield and sustainability on a global scale. It greatly reduces the land area that is planted and lowers crop production globally. In all living organisms, transcription factors (TFs) play a crucial role in regulating gene expression. They participate in cell signaling, cell cycle, development, and plant stress response. Plant resilience to diverse abiotic stressors is largely influenced by TFs. Transcription factors modulate gene expression by binding to their target gene's cis-elements, which are impacted by genomic characteristics, DNA structure, and TF interconnections. In this review, we focus onthe six major TFs implicated in abiotic stress tolerance, namely, DREB, bZIP, WRKY, ABF, MYB, and NAC, and the cruciality of phosphorylation of these transcription factors in abiotic stress signaling, as protein phosphorylation has emerged as one of the key post-translational modifications, playing a critical role in cell signaling, DNA amplification, gene expression and differentiation, and modification of other biological configurations. These TFs have been discovered after extensive study as stress-responsive transcription factors which may be major targets for crop development and important contributors to stress tolerance and crop production.

Role of Dof Transcription Factors under Abiotic Stresses

For the survival of the rapidly growing global populace, plant species must exhibit tolerance towards climate change. Plants possess mechanisms to respond to stress by changing their biological processes and stimulating stress-responsive genes. The Dof (TFs) family, which binds to DNA with a single finger, reflects a plant-specific group of TFs that play an important part in regulating plants that are facing different types of abiotic stresses which may influence their growth and development. Discovery of this family has made a significant impact on the field of plant sciences. However, the characterization of Dof transcription factors in crop plants is currently limited reported. Several Dof transcription factors (TFs) of plants have been shown in nature. The transcription factors TaDofs, StDof, MnDofs, JrDof3TF, Va/VvDofs, GhDof1, OsDof1, SmeDof, ZmDof, CsDof, DcDof, CaDofs, ThDof, BraDof, and AcDof are important for abiotic stressors such heat, cold, salt, drought, and heavy metals. In addition, Dof transcription factors play a role in the regulation of factors related to yield and quality. Nevertheless, some outstanding issues remain. The review article provides a summary of the role of various stress-responsive Dof transcription factors in response to abiotic stresses. Additionally, this study investigates the limitations and possible opportunities associated with Dof transcription factors in the development of crops that are capable of withstanding climate change. Therefore, it is recommended to conduct comprehensive research on Dof transcription factors (TFs) across many different transcription factors fields to find their potential novel functionality, which will be beneficial to our retention of the delicate biological processes in plants.

Transcription Factors in the Regulation of Plant Heat Responses

With the increasing global warming, heat stress has been a major challenge for plant growth and development. Transcriptional regulation is an important process in plant to combat heat stress. Recent studies have revealed the complicated transcriptional regulatory networks involved in plant heat stress response. Here, we review the latest advances regarding the transcriptional regulatory network and summarize the regulatory mechanisms of these heat stress-responsive transcription factors. We also explore the potential internal relationships among the major heat stress-responsive transcription factors. We believe that our knowledge on the regulatory mechanisms under plant heat stress will finally be transformed into crop plants for enhancing crop resistance and yields in future.

Genome-wide identification, molecular structures and functional exploration of the membrane attack complex/perforin domain-containing proteins and validation of GmmiRNA169o-GmMACPF-9 module in soybean cold stress

Soybean (Glycine max L.) is a widely cultivated legume and an enriched source of edible oil and protein. Membrane Attack Complex/Perforin Domain-Containing Proteins (MACPFs) played significant roles in plant growth and biotic and abiotic stresses. Here, we performed a genome-wide scanning for the putative GmMACPFs and a total of 20 GmMACPFs were identified in soybean. The conserved domain and phylogenetic analysis of MACPF showed the evolutionary diversification of MACPF genes family among legumes. The GmMACPFs protein characteristics were predicted, including theoretical molecular weight (38.06 to 68.38 kDa), protein isoelectric point (6.61 to 9.41), subcellular localization (nucleus, cytoplasm or mitochondria) and various TFs binding sites. GmMACPFs are located on 12 chromosomes out of a total 20 chromosomes in soybean. The 2 kb promoter analysis revealed that GmMACPFs contains a large number of phytohormones and stress responsive cis-acting elements (CAE) and transcription factor binding sites. We explored the 3D protein structure of GmMACPFs, their posttranscriptional regulators especially abiotic stress responsive miRNAs, and the evolutionary relationship among different legumes and Arabidopsis. GmMACPFs expression pattern was retrieved from RNA-seq libraries for tissue specific and under various abiotic stresses in soybean. According to the results, GmMACPF genes exhibited spatiotemporal, tissue, development stage and abiotic stress specific expression patterns in soybean. Our RT-qPCR analysis indicates the antagonistic expression pattern between GmmiRNA-GmMACPF modules under cold, salt, drought and heat stresses. Transient over expression of GmmiRNA169o-GmMACPF-9 module in soybean hairy root and biochemical analysis showed that GmmiRNA169o-OE plants accumulated less reactive oxygen species (ROS), malondialdehyde (MDA) and accumulated higher superoxide dismutase (SOD) and peroxidase (POD) to enhance cold stress tolerance. These results enriched our knowledge of the functions of GmmiRNA169o and GmMACPF-9 and provided candidate miRNAs-GmMACPFs modules especially GmmiRNA169o for environmental stress resilient soybean breeding through genetic engineering.

Succinylation of a KEAP1 sensor lysine promotes NRF2 activation

Cross talk between metabolism and stress-responsive signaling is essential for maintaining cellular homeostasis. This cross talk is often achieved through covalent modification of proteins by endogenous, reactive metabolites that regulate key stress-responsive transcription factors like NRF2. Metabolites including methylglyoxal, glyceraldehyde 3-phosphate, fumarate, and itaconate covalently modify sensor cysteines of the NRF2 repressor KEAP1, resulting in stabilization of NRF2 and activation of its cytoprotective transcriptional program. Here, we employed a shRNA-based screen targeting the enzymes of central carbon metabolism to identify additional regulatory nodes bridging metabolism to NRF2 activation. Succinic anhydride, increased by genetic depletion of the TCA cycle enzyme succinyl-CoA synthetase or by direct administration, results in N-succinylation of lysine 131 of KEAP1 to activate NRF2 signaling. This study identifies KEAP1 as capable of sensing reactive metabolites not only by several cysteine residues but also by a conserved lysine residue, indicating its potential to sense an expanded repertoire of reactive metabolic messengers.

Abstract PR010: In-“speck”-ting the nucleus: Nuclear speckles are a critical layer of gene regulation that predict outcomes in clear cell renal cell carcinoma

Abstract Nuclear speckles are dynamic substructures within the nucleus that contain a myriad of factors involved in gene activation. While nuclear speckles were discovered over 100 years ago, how they vary in human health and disease and the consequences of such variation remain enigmatic. Using RNA-seq data to estimate nuclear speckle content, we found that nuclear speckle phenotypes vary reproducibly in >20 cancer types. Of these cancers, nuclear speckle variation predicted patient outcomes specifically in clear cell renal cell carcinoma (ccRCC). This ccRCC speckle sensitivity was confirmed in a separate cohort using immunofluorescence to independently assess speckle phenotypes, and was specific to ccRCC with VHL mutation or copy number loss, suggesting involvement of the VHL-degraded HIF stress-responsive transcription factors. Given our previous findings linking nuclear speckles to another stress-responsive transcription factor, p53, we hypothesized that the speckle sensitivities we observed in ccRCC may be a consequence of as-of-yet uncharacterized speckle functions of HIF1A or HIF2A. Previously, we found that p53 drives DNA-speckle contacts of a subset of target genes to boost expression, revealing a novel method of transcription-factor-based gene regulation. However, the extent that DNA-speckle positioning is regulated by transcription factors other than p53, including the HIF transcription factors, is currently unclear. Critically, p53 and HIF2A, but not HIF1A, share a homologous protein motif, now coined the speckle targeting motif, that precisely overlaps with the region where we had mapped p53 DNA-speckle targeting abilities. Using genomic and imaging-based methods, we identified HIF2A as a second transcription factor that drives association between a subset of its target genes and nuclear speckles. Deletion of the HIF2A speckle targeting motifs ablated its DNA-speckle targeting functions, led to lower induction of speckle-associating genes, and resulted in a HIF2A-mediated gene expression program that more closely resembled the better-outcome ccRCC speckle patient group. Beyond p53 and HIF2A, we found that the speckle targeting motif occurs within hundreds of proteins and is specifically enriched among transcriptional regulators. These findings suggest that DNA-speckle targeting by transcription factors is potentially a wide-spread mechanism of gene regulation that may be manipulated by changing speckle-targeting abilities of transcription factors or by altering speckles themselves. For ccRCC, our findings reveal nuclear speckle phenotypes as novel prognostic indicators and illuminate nuclear speckles and HIF2A speckle-targeting abilities as new avenues for therapeutic development. Citation Format: Katherine A. Alexander, Ruofan Yu, Christine Faunce, Nicolas Skuli, Nathan Coffey, Son Nguyen, Nicholas Biddle, Catherine Li, Eric Joyce, Arjun Raj, M. Celeste Simon, Shelley Berger. In-“speck”-ting the nucleus: Nuclear speckles are a critical layer of gene regulation that predict outcomes in clear cell renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr PR010.

Analysis of an antioxidative defence system of hydrogen peroxide-treated pancreatic islet-derived 1.1B4 cells and siRNA targeting NR4A3-treated cells by microarray

ABSTRACTPancreatic islet β-cells weaken under oxidative stress. In this study, human pancreatic islet-derived 1.1B4 cells were exposed to H2O2 and analysed using a human microarray, which revealed that heme oxygenase 1 (HMOX1), glutamate-cysteine ligase, early growth response 1, nuclear receptor subfamily 4 group A member 3 (NR4A3) and jun B proto-oncogene were upregulated, whereas superoxide dismutase 1 and catalase were not. Expression of NR4A3 rapidly increased after H2O2 addition, and the 1.1B4 cells treated with siRNA targeting NR4A3 became sensitive to H2O2; further, HMOX1 expression was strongly inhibited, suggesting that NR4A3 is an oxidative stress-responsive transcription factor that functions through HMOX1 expression in pancreatic islet β-cells. Expression of cyclin E1 and cyclin-dependent kinase 1 was also inhibited by siRNAs targeting NR4A3.

Arabidopsis BBX14 negatively regulates nitrogen starvation- and dark-induced leaf senescence.

Senescence is a highly regulated process driven by developmental age and environmental factors. Although leaf senescence is accelerated by nitrogen (N) deficiency, the underlying physiological and molecular mechanisms are largely unknown. Here, we reveal that BBX14, a previously uncharacterized BBX-type transcription factor in Arabidopsis, is crucial for N starvation-induced leaf senescence. We find that inhibiting BBX14 by artificial miRNA (amiRNA) accelerates senescence during N starvation and in darkness, while BBX14 overexpression (BBX14-OX) delays it, identifying BBX14 as a negative regulator of N starvation- and dark-induced senescence. During N starvation, nitrate and amino acids like glutamic acid, glutamine, aspartic acid, and asparagine were highly retained in BBX14-OX leaves compared to the wild type. Transcriptome analysis showed a large number of senescence-associated genes (SAGs) to be differentially expressed between BBX14-OX and wild-type plants, including ETHYLENE INSENSITIVE3 (EIN3) which regulates N signaling and leaf senescence. Chromatin immunoprecipitation (ChIP) showed that BBX14 directly regulates EIN3 transcription. Furthermore, we revealed the upstream transcriptional cascade of BBX14. By yeast one-hybrid screen and ChIP, we found that MYB44, a stress-responsive MYB transcription factor, directly binds to the promoter of BBX14 and activates its expression. In addition, Phytochrome Interacting Factor 4 (PIF4) binds to the promoter of BBX14 to repress BBX14 transcription. Thus, BBX14 functions as a negative regulator of N starvation-induced senescence through EIN3 and is directly regulated by PIF4 and MYB44.

Pokkali: A Naturally Evolved Salt-Tolerant Rice Shows a Distinguished Set of lncRNAs Possibly Contributing to the Tolerant Phenotype.

Pokkali is a strong representation of how stress-tolerant genotypes have evolved due to natural selection pressure. Numerous omics-based investigations have indicated different categories of stress-related genes and proteins, possibly contributing to salinity tolerance in this wild rice. However, a comprehensive study towards understanding the role of long-noncoding RNAs (lncRNAs) in the salinity response of Pokkali has not been done to date. We have identified salt-responsive lncRNAs from contrasting rice genotypes IR64 and Pokkali. A total of 63 and 81 salinity-responsive lncRNAs were differentially expressed in IR64 and Pokkali, respectively. Molecular characterization of lncRNAs and lncRNA-miRNA-mRNA interaction networks helps to explore the role of lncRNAs in the stress response. Functional annotation revealed that identified lncRNAs modulate various cellular processes, including transcriptional regulation, ion homeostasis, and secondary metabolite production. Additionally, lncRNAs were predicted to bind stress-responsive transcription factors, namely ERF, DOF, and WRKY. In addition to salinity, expression profiling was also performed under other abiotic stresses and phytohormone treatments. A positive modulation in TCONS_00035411, TCONS_00059828, and TCONS_00096512 under both abiotic stress and phytohormone treatments could be considered as being of potential interest for the further functional characterization of IncRNA. Thus, extensive analysis of lncRNAs under various treatments helps to delineate stress tolerance mechanisms and possible cross-talk.

Genetic inactivation of essential HSF1 reveals an isolated transcriptional stress response selectively induced by protein misfolding.

Heat Shock Factor 1 (Hsf1) in yeast drives the basal transcription of key proteostasis factors and its activity is induced as part of the core heat shock response. Exploring Hsf1 specific functions has been challenging due to the essential nature of the HSF1 gene and the extensive overlap of target promoters with environmental stress response (ESR) transcription factors Msn2 and Msn4 (Msn2/4). In this study, we constructed a viable hsf1∆ strain by replacing the HSF1 open reading frame with genes that constitutively express Hsp40, Hsp70, and Hsp90 from Hsf1-independent promoters. Phenotypic analysis showed that the hsf1∆ strain grows slowly, is sensitive to heat as well as protein misfolding and accumulates protein aggregates. Transcriptome analysis revealed that the transcriptional response to protein misfolding induced by azetidine-2-carboxylic acid is fully dependent on Hsf1. In contrast, the hsf1∆ strain responded to heat shock through the ESR. Following HS, Hsf1 and Msn2/4 showed functional compensatory induction with stronger activation of the remaining stress pathway when the other branch was inactivated. Thus, we provide a long-overdue genetic test of the function of Hsf1 in yeast using the novel hsf1∆ construct. Our data highlight that the accumulation of misfolded proteins is uniquely sensed by Hsf1-Hsp70 chaperone titration inducing a highly selective transcriptional stress response.

The Nuclear Localization Signal of NF-κB p50 Enters the Cells via Syndecan-Mediated Endocytosis and Inhibits NF-κB Activity

It is well established that cationic peptides can enter cells following attachment to polyanionic membrane components. We report that the basic nuclear localization signal (NLS) of the NF-κB p50 subunit is internalized via lipid raft-dependent endocytosis mediated by heparan sulfate proteoglycans and exerts significant NF-κB inhibitory activities both in vitro and in vivo. In vitro uptake experiments revealed that the p50 NLS peptide (CYVQRKRQKLMP) enters the cytoplasm and accumulates in the nucleus at 37 °C. Depleting cellular ATP pools or decreasing temperature to 4 °C abolished peptide internalization, confirming the active, energy-dependent endocytic uptake. Co-incubation with heparan sulfate or replacing the peptide’s basic residues with glycines markedly reduced the intracellular entry of the p50 NLS, referring to the role of polyanionic cell-surface proteoglycans in internalization. Furthermore, treatment with methyl-β-cyclodextrin greatly inhibited the peptide’s membrane translocation. Overexpression of the isoforms of the syndecan family of transmembrane proteoglycans, especially syndecan-4, increased the cellular internalization of the NLS, suggesting syndecans’ involvement in the peptide’s cellular uptake. In vitro, p50 NLS reduced NF-κB activity in TNF-α-induced L929 fibroblasts and LPS-stimulated RAW 264.7 macrophages. TNF-α-induced ICAM-1 expression of HMEC-1 human endothelial cells could also be inhibited by the peptide. Fifteen minutes after its intraperitoneal injection, the peptide rapidly entered the cells of the pancreas, an organ with marked syndecan-4 expression. In an acute pancreatitis model, an inflammatory disorder triggered by the activation of stress-responsive transcription factors like NF-κB, administration of the p50 NLS peptide reduced the severity of pancreatic inflammation by blocking NF-κB transcription activity and ameliorating the examined laboratory and histological markers of pancreatitis.