Search Results (672)

Fzf1 is a Saccharomyces cerevisiae transcription factor that contains five zinc finger domains (ZF1-5) and induces the expression of at least five genes in response to various chemical stresses by recognizing the shared promoter consensus sequence CS2. The N-terminal ZF1-3 are required and sufficient for binding to CS2, while ZF4 negatively regulates the activity of Fzf1. However, the effect of ZF5 on the activity of Fzf1 is not well defined. In this study, substitutions of the two zinc-coordinating Cys residues (C248S and C253S) of ZF5, or deletion of the whole ZF5 domain, compromised the chemical stress-induced activation of Fzf1. Since the elevated Fzf1-regulated gene expression caused by fzf1-ZF4 could also be reversed by additional deletion of ZF5 or C248S/C253S substitutions, fzf1-ZF5 mutations are epistatic over fzf1-ZF4 mutations. Furthermore, fzf1-ZF5 mutations are recessive to FZF1, while ZF5 is dispensable for the CS2 binding. Finally, Fzf1-ZF5 is required and sufficient to serve as a transcription activation domain when fused to a Gal4 DNA-binding domain. These observations collectively support a working model in which Fzf1 bound to its target gene promoters remains inactive due to an inhibitory activity of ZF4. Upon chemical stress, ZF4 is no longer able to inhibit the ZF5 transactivation activity, leading to the induction of Fzf1-regulated gene expression and subsequent chemical detoxification. Full article

Background/Objectives: Atrial fibrillation (AF), characteristic of chaotic atrial electrical activity along with ineffective atrial systole, remains the most frequent sustained cardiac dysrhythmia, with an overall lifetime risk for AF being approximately 15% to 40% in the global population. AF is associated with substantially enhanced risks for multiple adverse clinical outcomes, including thromboembolic cerebral stroke, dementia, chronic kidney disease, myocardial infarction, cardiac failure, and even premature cardiac demise. Although remarkable advances have been achieved toward unravelling the complex hereditary etiopathogenesis underpinning AF, it has become increasingly clear that inherited determinants predisposing to AF in a vast majority of individuals are still uncertain. Methods: A Chinese pedigree with idiopathic AF and another group of 236 cases suffering idiopathic AF along with 312 unrelated healthy volunteers were prospectively recruited. Exome-wide sequencing and Sanger sequencing assays were implemented in research participants. The functional effects of the discovered variations in the SOX5 gene were explored through dual-luciferase reporter analysis. Results: Two novel SOX5 mutants, NM_006940.6: c.355C>T; p.(Gln119*) and NM_006940.6: c.640G>T; p.(Glu214*), were identified in the AF pedigree and one of the 236 unrelated patients affected with AF, respectively. These two heterozygous truncating SOX5 variations were absent from the 624 control chromosomes. Quantitative luciferase reporter assays unraveled that both Gln119*- and Glu214*-mutant SOX5 lost the ability to transactivate GJA1. Additionally, the two variations abolished the synergistic transactivation of SCN5A by SOX5 and SHOX2. Conclusions: The current findings indicate SOX5 as a novel gene contributing to AF, which adds more insight to the molecular pathogenesis of AF, and provides a potential target for personalized precision medicine. Full article

Mutations in the POU1F1 gene cause defects in the expression of the genes encoding thyroid-stimulating hormone (TSH)-β subunit, growth hormone (GH), and prolactin (PRL). Here, we characterized 15 missense and nonsense mutations. Protein stability was reduced in the P14L, P24L, F135C, K145X, F233S and E250X mutants. Transactivation by 15 mutants in the TSHβ promoter was moderately correlated with that of the PRL promoter. Based on their transcriptional activity, we classified them into three groups: group I, equivalent to the wild type; group II, partial; and group III, substantially lost. A review of case reports on four patients with group II mutations revealed that TSH deficiency manifested after recombinant GH therapy. A transcription factor, GATA2, is the main activator in the TSHβ gene, while POU1F1 protects its function from inhibition by the suppressor region (SR). We found that the SR is critical for the pathogenesis of TSH deficiency. The transactivation of the TSHβ promoter by the K216E mutant was equivalent to that of wild-type POU1F1; however, that of the PRL promoter was low, while the opposite was found in the R271W mutant. The functional property of K216E suggests that the interaction of POU1F1 with GATA2 may not always be necessary for the activation of the TSHβ promoter. Full article

Actinidia valvata, a promising rootstock for kiwifruit cultivation, demonstrates superior waterlogging tolerance compared with commercial cultivars. Lateral organ boundaries domain (LBD) transcription factors (TFs) are known to be pivotal in plant responses to abiotic stress. Nevertheless, the characterization of the LBD family under waterlogging stress in A. valvata remains limited. In this study, 26 AvLBD genes were identified from a transcriptome dataset, with the majority classified into phylogenetic Class II. Under waterlogging stress, transcript accumulation of most AvLBD41 members, particularly AvLBD41_11 and AvLBD41_7, was markedly increased in roots. Bimolecular fluorescence complementation (BiFC) assays indicated that AvLBD41_7 heterodimerizes with both the AP2/ERF activator AvERF75 and the trihelix repressor AvHRA1, whereas AvLBD41_11 only interacts with AvERF75. Neither AvLBD41 isoform interacts with AvERF73, thereby defining distinct components of a waterlogging-responsive module. Yeast-based assays revealed an absence of transactivation activity for AvLBD41_7, and transient expression analyses confirmed its exclusive nuclear localization. The promoters of both AvLBD41_11 and AvLBD41_7 harbor numerous cis-elements responsive to hormones and abiotic stresses. An AvLBD41_7-derived PCR marker could be used to distinguish A. valvata from A. deliciosa accessions. Collectively, these findings provide a comprehensive functional annotation of the LBD gene family in A. valvata and establish AvLBD41_7 as a potential molecular target for future kiwifruit breeding programs aimed at waterlogging resilience. Full article

Nucleopeptides (NPs) are unnatural hybrid polymers designed by coupling nucleobases to the side chains of amino acid residues within peptides. In this study, we present the synthesis of an Fmoc-protected nucleobase amino acid (NBA) monomer (Fmoc-1,4-TzlNBA_A) with adenine attached to the side chain of L-homoazidoalanine (Aha) through a 1,4-linked-1,2,3-triazole. The coupling was accomplished by a Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) of Fmoc-Aha and N9-propargyladenine. Subsequently, a homotrinucleopeptide (HalTzl_AAA) containing three 1,4-TzlNBA_A residues was synthesized, using different solid-phase peptide synthesis (SPPS) approaches, and its ability to recognize U-rich motifs of RNAs involved in the HIV replication cycle was studied using circular dichroism (CD) and fluorescence spectroscopy. CD curves confirmed the binding of HalTzl_AAA to U-rich motifs of the transactivation responsive element (TAR UUU RNA HIV-1) bulge and the anticodon stem–loop domain of human tRNALys3 (ASLLys3) by a decrease in the positive ellipticity band intensity around 265 nm during the complexation. 5′-(FAM(6))-labeled TAR UUU and hASLLys3 were used for fluorescence anisotropy binding studies. Fluorescence data revealed that HalTzl_AAA bound TAR’s UUU bulge with a moderate affinity (K_d ≈ 38 µM), whereas the ASLLys3 UUUU-containing loop sequence was recognized with 2.5 times lower affinity (with K_d ≈ 75 µM). Both the standard SPPS method and its variants, which involved the attachment of adenine to the L-Aha side chain using the click reaction during the synthesis on the resin or after the nucleopeptide cleavage, were characterized by a similar efficiency and yield. The CD and fluorescence results demonstrated that HalTzl_AAA recognized the U-rich sequences of the RNAs with moderate and varied affinities. It is likely that both the hydrogen bonds associated with the complementarity of the interacting sequences and the conformational aspects associated with the high conformational dynamics of U-rich motifs are important in the recognition process. The nucleopeptide represents a new class of RNA binders and may be a promising scaffold for the development of new antiviral drugs. Full article

The Krüppel-like transcription factor GLIS3 plays an important regulatory role in the development of various tissues, both in mice and humans. Loss-of-function mutations in GLIS3 are implicated in several pathologies, including polycystic kidney disease, diabetes, and hypothyroidism. Previous studies have reported that the mouse Glis3 gene generates a 7524 bp mRNA encoding a 935 amino acid (aa) protein, with a homologous human protein of 930 aa. Here, we identify a shorter mouse mRNA lacking the third exon, producing a shorter 659 aa GLIS3 protein. This shorter transcript is expressed at a higher level than the longer transcript in all mouse tissues tested and produces a protein that is more stable and exhibits a greater transactivation potential. This suggests that the 276 aa N-terminus in the longer mouse GLIS3 protein encompasses important regulatory domain(s). Mass spectrometry identified several phosphorylation sites that may contribute to the post-translational regulation of GLIS3 activity and function and several known members of co-activator and co-repressor complexes, consistent with the concept that GLIS3 can act both as a transcriptional repressor and activator. These data offer important insights into how GLIS3 activity is regulated and offer potential mechanisms for its control during tissue development and disease. Full article

Background/Objectives: Rosiglitazone (RSG), a potent PPARγ agonist for type 2 diabetes mellitus (T2DM), induces adverse adipogenic effects that limit clinical use. We investigated whether emodin (1,3,8-trihydroxy-6-methylanthraquinone, EMO), a natural anthraquinone, mitigates RSG-induced complications while enhancing its insulin-sensitizing benefits in severe obesity. Methods: Male ob/ob mice with established obesity and diabetes were treated for 4 weeks with RSG (10 mg kg⁻¹ day⁻¹), EMO (200 or 400 mg kg⁻¹ day⁻¹) or their combination. Metabolic profiling, organ function, and adipose histology were analyzed. RNA sequencing and mechanistic studies (Western blot, RT-qPCR, luciferase assays) in inguinal subcutaneous adipose tissue (iSAT), epididymal white adipose tissue (eWAT), and 3T3-L1 adipocytes were used to define EMO’s actions. Results: EMO co-treatment dose-dependently reduced RSG-induced weight gain, visceral adiposity (iSAT and eWAT mass, p < 0.05), and ectopic lipid deposition while ameliorating hepatorenal dysfunction. EMO synergistically enhanced RSG’s glucose-lowering effects. Mechanistically, EMO suppressed sterol regulatory element-binding protein 1 (SREBP1)-mediated lipogenesis (Srebp1, Acc, Fasn, Scd1; p < 0.05) and enhanced PPARγ-peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α)-driven thermogenesis via enhanced PPARγ transactivation and nuclear translocation. Thermogenic genes (Ucp1, Ppargc1a, Cidea; p < 0.05) were upregulated, with maximal uncoupling protein 1 (UCP1) induction in iSAT at 400 mg/kg EMO. Conclusions: EMO selectively enhances RSG’s glycemic benefits while attenuating its adipogenic effects in severe obesity by dual PPARγ modulation-inhibiting adipogenic pathways while amplifying thermogenesis. This strategy mitigates RSG’s adverse effects while improving insulin sensitivity, supporting the potential of EMO as a PPARγ adjunct therapy. Full article

Glucocorticoids (GCs) represent effective anti-cancer drugs for the treatment of hematological malignancies, but their clinical use is limited due to their multiple adverse effects. Selective glucocorticoid receptor agonists/modulators (SEGRAMs) modify glucocorticoid receptor (GR) function, shifting it towards therapeutically important transrepression and, therefore, could be safer alternative to GCs. Here we report on the biological activity of four novel glucocorticoid receptor (GR) ligands, derivatives of synephrine, a natural-origin molecule. We demonstrated the affinity of synephrine derivatives in silico and in vitro by molecular dynamics simulation and radioligand binding assay, correspondingly. Further, we tested the induction of apoptosis in cultured cells and cytotoxic effects in primary lymphoblasts from patients with acute lymphoblastic leukemia. Therapeutically important GR transrepression was evaluated by luciferase reporter assay and Q-PCR of transrepression marker genes, while GR transactivation associated with side effects was evaluated by Q-PCR analysis and by the level of GR phosphorylation at Ser211. Anti-cancer effects of the leader compound, 1-[4-(benzyloxy)phenyl]-2-(hexylamino)ethanol (10S-E2), were studied using a murine transplantable lymphoma P388 model. The potential of 10S-E2 to prevent the development of atrophic complication was evaluated using a murine model of glucocorticoid-induced osteoporosis. All studied synephrine derivatives demonstrated high GR affinity, with the IC₅₀ value of the most active derivative 10S-E2 being 0.56 µM; the effects on GR function were cell-type-specific. The leader compound, 10S-E2, revealed SEGRAM properties in vitro and demonstrated anti-cancer effects in vivo, inhibiting tumor growth by more than 60%. Although the anti-cancer effect of 10S-E2 was less pronounced than that of the reference drug dexamethasone, non-atrophogenic properties of 10S-E2 make this molecule an attractive candidate for long-term GR-associated therapies. Full article

The HIV-1 transactivator of transcription (TAT) protein enhances beta amyloid (Aβ42) neurotoxicity and may accelerate Alzheimer’s disease (AD)-related neuronal damage, yet its impact on nuclear architecture remains unclear. In this study, we examined the mechanism by which TAT–Aβ42 affects nuclear integrity. Exposure to TAT–Aβ42-induced a marked elevation in intracellular calcium levels, which subsequently activated cathepsin L (CL), a lysosomal cysteine protease. Activated CL cleaved nuclear lamins, leading to nuclear envelope disruption and altered nuclear morphology. Both calcium chelation and pharmacological inhibition of CL significantly reduced lamin cleavage, highlighting a calcium-dependent CL-mediated pathway. These findings identify a novel mechanism by which TAT–Aβ42 compromises nuclear architecture, providing mechanistic insight into how HIV infection may exacerbate neurodegenerative processes in AD. Full article

Bovine viral diarrhea virus (BVDV) is a major pathogen responsible for significant economic losses in the global cattle industry. The diverse transmission routes and the characteristics of asymptomatic infections make it difficult to contain the spread; there is an urgent need to develop new effective antiviral strategies. Nanobodies (Nbs) have become a promising new type of antiviral agent due to their advantages, including small molecular size, stable structure, high specificity, and ease of production. This study successfully screened a specific nanobody, Nb7, targeting the key functional protein NS5A of BVDV using phage display technology. Furthermore, the nanobody was effectively delivered into Madin–Darby bovine kidney (MDBK) cells by fusing it with the cell-penetrating peptide TAT. The results demonstrate that TAT-Nb7, specifically targeting the non-structural protein NS5A of BVDV, significantly inhibits viral replication in MDBK cells. In conclusion, this study indicates that TAT-Nb7 holds promise as a therapeutic candidate for the prevention and control of BVDV infection. Full article

Soil salinization significantly limits plant growth and agricultural productivity, with MYB transcription factors playing crucial roles in mediating plant responses to salt stress. In this study, a novel R2R3-MYB transcription factor gene, SlMYB306-like, was isolated from tomato. Phylogenetic comparison indicated that SlMYB306-like shared the highest sequence homology with potato StMYB306-like. Subcellular localization assays demonstrated nuclear localization of SlMYB306-like protein, while yeast transactivation assays confirmed its function as a transcriptional activator. Expression profiling showed that SlMYB306-like was inducible by NaCl and abscisic acid (ABA) treatments. In addition, functional characterization via the overexpression of SlMYB306-like in Arabidopsis thaliana revealed enhanced salt tolerance, evidenced by an increased maximum quantum efficiency of photosystem II (Fv/Fm) and proline levels alongside decreased accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) content under salt stress conditions. Furthermore, the overexpression of SlMYB306-like upregulated the expression of several stress-responsive genes, including AtSOD1, AtCAT1, AtEGY3, AtP5CS2, and AtRD29A. Collectively, these findings suggest that SlMYB306-like enhances salt tolerance by modulating ROS scavenging, osmotic adjustment, and ABA signaling pathways, thereby representing a promising candidate gene for the development of salt-tolerant crops. Full article

Glucocorticoids are essential for fetal organ maturation and form the basis of antenatal corticosteroid therapy that has significantly reduced preterm-related morbidity such as respiratory distress syndrome (RDS). However, neonatal morbidity remains a clinical challenge regardless of antenatal corticosteroid therapy. Currently, it is thought that adverse intrauterine environments dysregulate glucocorticoid receptor (GR) homeostasis, yet the biological mechanisms remain poorly understood. Therefore, we aimed to study ex vivo glucocorticoid sensitivity in cord blood immune cells from two independent preterm cohorts to identify associations with neonatal morbidity and uncover potential mechanisms of dysregulated glucocorticoid homeostasis. In the first cohort, thawed cord blood mononuclear cells were exposed to betamethasone in the presence of lipopolysaccharides (LPS) for 4 h. In the second cohort, freshly isolated white blood cells were treated with dexamethasone under unstimulated and LPS-stimulated conditions for 48 h. GR isoform expression and regulation of transactivated and transrepressed genes were assessed via qPCR, immunoblotting, flow cytometry, and ELISA. In both cohorts, reduced GR expression, particularly of the GRα isoform, was observed in neonates with morbidity, but only with culture time and not in freshly isolated cells. Ex vivo impaired glucocorticoid-mediated transrepression of proinflammatory genes IL6 and TNF was also observed in the morbidity groups. In contrast, all samples were comparable in basal immune cell distributions and transactivation of glucocorticoid response element (GRE)-dependent genes GILZ and FKBP5, irrespective of neonatal morbidity. These findings suggest that neonates that develop morbidities experience an early postnatal GR dysfunction that is potentially programmed in utero. Moreover, under conditions of decreased GR abundance, classical transactivation functions appear to be preserved at the expense of more complex regulatory mechanisms such as transrepression. Full article

Drought stress is a major limiting factor during the process of plant growth and development, especially in arid and semi-arid regions. MYB transcription factors play vital roles in the regulation of many developmental processes under various stresses. The aim of this study was to determine whether PtrMYB119 enhanced dehydration tolerance in Nicotiana tabacum. PtrMYB119, with a weak transactivation activity, was distributed throughout the cell with no apparent specificity. The transgenic tobacco overexpressing PtrMYB119 might regulate dehydration tolerance through increased ABA content and antioxidant enzyme activities, decreased MDA levels, and up-regulation of antioxidant genes, polyamine biosynthesis genes, and drought-responsive genes. Overall, our results could contribute to the elucidation of drought tolerance underlying PtrMYB119 action in tobacco and indicated that PtrMYB119 could be exploited for engineering drought-enduring plants in the future. Full article

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL). The trans-activator protein Tax of HTLV-1 is thought to play a crucial role in the early-stage transformation of the virus-infected cells. Tax is a multi-functional protein and modulates cellular signaling pathways that promote proliferation and survival of HTLV-1-infected cells, primarily through the trans-activation of cellular target genes. Tax interacts with a variety of host cell factors including signal transducers and transcription factors, leading to the activation of transcription factors such as CREB, NF-κB, and SRF and activates both its own promoter and those of a variety of host cellular genes. Tax activates its own promoter mainly through CREB and host cellular genes through NF-κB, SRF, and CREB. Accumulating evidence indicates that the Tax-mediated trans-activation of target genes through NF-κB plays an essential role in the transformation of HTLV-1 infected cells. However, the repertoire of Tax target genes, especially those crucial for leukemogenesis, are not known in detail. In this review, we summarize transcriptional activation mechanisms and target genes of Tax, especially focusing on transformation, to facilitate understanding of the underlying mechanisms of leukemogenesis induced by HTLV-1 infection. Full article

Miscanthus, a perennial grass, is renowned for its remarkable tolerance to abiotic stress. Excessive levels of drought severely impair plant growth and yield. Plant nuclear factor Y (NF-Y) transcription factors (TFs) play pivotal roles in regulating responses to drought stress in species such as Arabidopsis and maize. However, their functional roles in conferring drought tolerance in Miscanthus remain largely unexplored. This study’s genome-wide analysis and gene expression profiling of Miscanthus under dehydration/osmotic stress identified a transcription factors gene, MsNF-YA4, which was significantly upregulated under dehydration/osmotic stress. MsNF-YA4 overexpression in Arabidopsis significantly enhanced drought tolerance, leading to increased transcription of stress- and antioxidant enzyme-related genes. Compared with the wild type (WT), the transgenic lines exhibited markedly higher relative water content (RWC), chlorophyll content, proline level, and antioxidant enzyme activity. Furthermore, the MsNF-YA4/MsNF-YB3/MsNF-YC2 improved the transactivation of the Miscanthus P5CS1, SOD (Cu/Zn) and CAT1 promoters in the transient system. These results offer fresh perspectives on the role of Miscanthus NF-YAs in drought tolerance and offer promising genetic resources for developing drought-tolerant crops through breeding programs. Full article