Nuclear Coactivator Research Articles

Tape-assisted fabrication method for constructing PDMS membrane-containing culture devices with cyclic radial stretching stimulation.

Advanced in vitro culture systems have emerged as alternatives to animal testing and traditional cell culture methods in biomedical research. Polydimethylsiloxane (PDMS) is frequently used in creating sophisticated culture devices owing to its elastomeric properties, which allow mechanical stretching to simulate physiological movements in cell experiments. We introduce a straightforward method that uses three types of commercial tape-generic, magic and masking-to fabricate PDMS membranes with microscale thicknesses (47.2 µm for generic, 58.1 µm for magic and 89.37 µm for masking) in these devices. These membranes are shaped as the bases of culture wells and can perform cyclic radial movements controlled via a vacuum system. In experiments with A549 cells under three mechanical stimulation conditions, we analysed transcriptional regulators responsive to external mechanical stimuli. Results indicated increased nuclear yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) activity in both confluent and densely packed cells under cyclically mechanical strains (Pearson's coefficient (PC) of 0.59 in confluent and 0.24 in dense cells) compared with static (PC = 0.47 in confluent and 0.13 in dense) and stretched conditions (PC = 0.55 in confluent and 0.20 in dense). This technique offers laboratories without microfabrication capabilities a viable option for exploring cellular behaviour under dynamic mechanical stimulation using PDMS membrane-equipped devices.

Charge Relaying within a Phospho-Motif Rescue Binding Competency of a Disordered Transcription Factor.

Structural disorder in proteins is central to cellular signaling, where conformational plasticity equips molecules to promiscuously interact with different partners. By engaging with multiple binding partners via the rearrangement of its three helices, the nuclear coactivator binding domain (NCBD) of the CBP/p300 transcription factor is a paradigmatic example of promiscuity. Recently, molecular simulations and experiments revealed that, through the establishment of long-range electrostatic interactions, intended as salt-bridges formed between the post-translationally inserted phosphate and positively charged residues in helix H3 of NCBD, phosphorylation triggers NCBD compaction, lowering its affinity for binding partners. By means of extensive molecular simulations, we here investigated the effect of short-range electrostatics on the conformational ensemble of NCBD, by monitoring the interactions between a phosphorylated serine and conserved positively charged residues within the NCBD phospho-motif. We found that empowering proximal electrostatic interactions, as opposed to long-range electrostatics, can reshape the NCBD ensemble rescuing the binding competency of phosphorylated NCBD. Given the conservation of positive charges in phospho-motifs, proximal electrostatic interactions might dampen the effects of phosphorylation and act as a relay to regulate phosphorylated intrinsically disordered proteins, ultimately tuning the binding affinity for different cellular partners.

Targeting Nuclear Receptor Coactivator SRC-1 Prevents Colorectal Cancer Immune Escape by Reducing Transcription and Protein Stability of PD-L1.

Programmed death-ligand 1 (PD-L1) is overexpressed in multiple cancers and critical for their immune escape. It has previously shown that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability, yet its role in CRC immune escape is unclear. Here, we demonstrate that SRC-1 is positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibits PD-L1 expression in CRC cells and retards murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration of CD8+ T cells. Genetic ablation of SRC-1 in mice also decreases PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, these findings highlight a crucial role of SRC-1 in regulating PD-L1 expression and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment.

Pygo-F773W Mutation Reveals Novel Functions beyond Wnt Signaling in Drosophila.

Pygopus (Pygo) has been identified as a specific nuclear co-activator of the canonical Wingless (Wg)/Wnt signaling pathway in Drosophila melanogaster. Pygo proteins consist of two conserved domains: an N-terminal homologous domain (NHD) and a C-terminal plant homologous domain (PHD). The PHD's ability to bind to di- and trimethylated lysine 4 of histone H3 (H3K4me2/3) appears to be independent of Wnt signaling. There is ongoing debate regarding the significance of Pygo's histone-binding capacity. Drosophila Pygo orthologs have a tryptophan (W) > phenylalanine (F) substitution in their histone pocket-divider compared to vertebrates, leading to reduced histone affinity. In this research, we utilized CRISPR/Cas9 technology to introduce the Pygo-F773W point mutation in Drosophila, successfully establishing a viable homozygous Pygo mutant line for the first time. Adult mutant flies displayed noticeable abnormalities in reproduction, locomotion, heart function, and lifespan. RNA-seq and cluster analysis indicated that the mutation primarily affected pathways related to immunity, metabolism, and posttranslational modification in adult flies rather than the Wnt signaling pathway. Additionally, a reduction in H3K9 acetylation levels during the embryonic stage was observed in the mutant strains. These findings support the notion that Pygo plays a wider role in chromatin remodeling, with its involvement in Wnt signaling representing only a specific aspect of its chromatin-related functions.

Abstract 1688: Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability

Abstract Programmed death-ligand 1 (PD-L1), one of the immunosuppressive molecules, overexpresses in multiple cancers and is critical for their immune escape. We previously showed that the nuclear coactivator SRC-1 promoted colorectal cancer (CRC) progression by enhancing CRC cell viability; however, the role of SRC-1 in CRC immune escape is unclear. Here, we demonstrated that SRC-1 was positively correlated with PD-L1 in human CRC specimens. SRC-1 deficiency significantly inhibited PD-L1 expression in both human and murine CRC cells and retarded murine CRC growth in subcutaneous grafts by enhancing CRC immune escape via increasing tumor infiltration and antitumor activity of effector CD8+ T cells. Genetic ablation of SRC-1 in mice also decreased PD-L1 expression in AOM/DSS-induced murine CRC. These results suggest that tumor-derived SRC-1 promotes CRC immune escape by enhancing PD-L1 expression. Mechanistically, SRC-1 activated JAK-STAT signaling by inhibiting SOCS1 expression and coactivated STAT3 and IRF1 to enhance PD-L1 transcription as well as stabilized PD-L1 protein by inhibiting proteasome-dependent degradation mediated by speckle type POZ protein (SPOP). Pharmacological inhibition of SRC-1 improved the antitumor effect of PD-L1 antibody in both subcutaneous graft and AOM/DSS-induced murine CRC models. Taken together, our findings highlight a crucial role of SRC-1 in facilitating CRC immune escape and targeting SRC-1 in combination with PD-L1 antibody immunotherapy may be an attractive strategy for CRC treatment. Citation Format: Yilin Hong, Qiang Chen, Zinan Wang, Yong Zhang, Bei Li, Xu Kong, Pingli Mo, Nengming Xiao, Jianming Xu, Yunbin Ye, Chundong Yu. Nuclear receptor coactivator SRC-1 promotes colorectal cancer immune escape by enhancing PD-L1 transcription and protein stability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1688.

Andrographolide attenuated MCT-induced HSOS via regulating NRF2-initiated mitochondrial biogenesis and antioxidant response.

Hepatic sinusoidal obstruction syndrome (HSOS) is a death-dealing liver disease with a fatality rate of up to 67%. In the study present, we explored the efficacy of andrographolide (Andro), a diterpene lactone from Andrographis Herba, in ameliorating the monocrotaline (MCT)-induced HSOS and the underlying mechanism. The alleviation of Andro on MCT-induced rats HSOS was proved by biochemical index detection, electron microscope observation, and liver histological evaluation. Detection of hepatic ATP content, mitochondrial DNA (mtDNA) copy number, and protein expression of nuclear respiratory factor-1 (NRF1) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) demonstrated that Andro strengthened mitochondrial biogenesis in livers from MCT-treated rats. Chromatin immunoprecipitation assay exhibited that Andro enhanced the occupation of nuclear factor erythroid 2-related factor 2 (NFE2L2, also known as NRF2) in the promoter regions of both PPARGC1A and NRF1. Andro also activated the NRF2-dependent anti-oxidative response and alleviated liver oxidative injury. In Nrf2 knock-out mice, MCT induced more severe liver damage, and Andro showed no alleviation in it. Furthermore, the Andro-activated mitochondrial biogenesis and anti-oxidative response were reduced in Nrf2 knock-out mice. Contrastingly, knocking out Kelch-like ECH-associated protein 1 (Keap1), a NRF2 repressor, reduced MCT-induced liver damage. Results from co-immunoprecipitation, molecular docking analysis, biotin-Andro pull-down, cellular thermal shift assay, and surface plasmon resonance assay showed that Andro hindered the NRF2-KEAP1 interaction via directly binding to KEAP1. In conclusion, our results revealed that NRF2-dependent liver mitochondrial biogenesis and anti-oxidative response were essential for the Andro-provided alleviation of the MCT-induced HSOS. Graphical Headlights: 1. Andro alleviated MCT-induced HSOS via activating antioxidative response and promoting mitochondrial biogenesis. 2. Andro-activated antioxidative response and mitochondrial biogenesis were NRF2-dependent. 3. Andro activated NRF2 via binding to KEAP1.

Dynamic observation of circRNA and mRNA profiles in a rat model of deep vein thrombosis.

The goal of the present study was to identify different transcriptome expression profiles involved in the pathogenesis of deep vein thrombosis (DVT), and to illustrate the diagnostic and therapeutic potential of circular RNAs (circRNAs) and mRNAs in DVT progression. A Sprague-Dawley rat model of DVT was successfully established through the stenosis method and samples were sequenced at four time points (1, 6 and 12 h, and 3 days after ligation) to observe the dynamic changes in circRNAs and mRNAs during DVT progression. RNA sequencing was used to analyze the circRNA and mRNA expression profiles, and associated functions and pathways, in the blood of DVT rats at the four time points. In addition, Short Time Series Expression Miner (STEM) analysis was performed to explore temporal gene expression. Differential expression of 1,680, 4,018, 3,724, and 3,036 circRNAs, and 400, 1,176, 373, and 573 mRNAs was observed in the 1, 6 and 12 h, and 3-day groups, respectively, compared with the sham group (fold change >2.0 or <-2.0, P<0.05). Functional enrichment analysis indicated that differentially expressed mRNAs were associated with the following terms: Immune response, cell activation, blood stasis facilitated organelle, extracellular membrane-bounded organelle, and blood microparticle, oxygen transporter activity. STEM analysis indicated that the expression of 366 circRNAs in circRNA profile 45 and 270 mRNAs in mRNA profile 45 was consistent with thrombus progression. Enrichment analysis was performed on mRNA profile 45. The main Gene Ontology annotations were chromosome segregation, mitotic sister chromatid segregation, cell cycle process, and ligand-dependent nuclear receptor transcription coactivator activity. Pathway enrichment analysis identified the platelet-associated pathway, immune-associated pathway, and inflammation-relation pathway. According to the enriched platelet-associated pathways, four mRNAs and ten candidate circRNAs were selected for reverse transcription-quantitative PCR verification. The expression of nine of the ten circRNAs and all four mRNAs was consistent with the sequencing results. In summary, differentially expressed circRNAs and mRNAs are dynamically involved in DVT development. Dysregulated transcriptome profiles and the corresponding functions and pathways may provide mechanistic insights into DVT diagnosis and treatment.

UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability

Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.

Estrogens receptors, nuclear coactivator 1 and ligand-dependent corepressor expression are altered early during induced ovarian follicular persistence in dairy cattle

Failure of ovulation can lead to follicular persistence, one of the main components of the pathogenesis of cystic ovarian disease (COD) in dairy cattle. Follicular persistence causes the permanence of a functional follicular structure in the ovary, which alters the cyclicity of the female and causes infertility. The aim of the present study was to evaluate the expression of estrogen receptors (ESR) 1 and 2, and the coregulatory proteins NCOA1, NRIP1 and LCOR by immunohistochemistry, in antral and preovulatory/persistent follicles in a model of follicular persistence induced by low levels of progesterone, to detect incipient changes during COD development, on the expected day of ovulation (P0) and after 5 (P5), 10 (P10) and 15 (P15) days of follicular persistence. Twenty-five Holstein cows were used, which were distributed in 5 groups: control group (n = 5), group P0 (n = 5), group P5 (n = 5), group P10 (n = 5), group P15 (n = 5). ESR1 expression was lower in antral follicles of the P5 (theca), P10 and P15 (theca and granulosa) groups relative to the control group (p < 0.05), and also lower in granulosa cells of persistent follicles of the P5, P10 and P15 groups than in dominant follicles of the control group (p < 0.05), without differences in theca cells. ESR2 expression showed no differences between groups. The ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as from 5 days of persistence (p < 0.05). NCOA1 expression was higher in granulosa cells of both antral and persistent follicles from the P0 group relative to the P5 and P10 groups, but showed no differences with the control and P15 groups (p < 0.05). Theca cells of antral and persistent follicles showed higher expression in the P0 and P15 groups in relation to the control, P5 and P10 groups (p < 0.05). No differences were detected for NRIP1 in antral, dominant and persistent follicles between groups. LCOR expression showed a decrease in granulosa cells of antral follicles from all persistence groups relative to the control group (p < 0.05). In theca cells, antral follicles of the P10 group showed lower LCOR expression than the control group (p < 0.05). LCOR expression was similar for dominant and persistent follicles. Considering that the ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as well as the decreased LCOR and NCOA1 expression, we may assume that, at the early stages of persistence, there is a negative regulation of ESR transcription. This coincides with the effects of estrogens through ESR on proliferation and apoptosis among other processes that favor follicular persistence. The results obtained provide relevant information in the knowledge of local events during the development of follicular persistence that could explain the failures in the reversion of the disease through hormonal treatments and the high recurrence rates reported for COD. In addition, it contributes to the study and identification of possible therapeutic targets, for the design of new treatments.

Lipophilic Statins Inhibit YAP Nuclear Localization, Coactivator Activity, and Migration in Response to Ligation of HLA Class I Molecules in Endothelial Cells: Role of YAP Multisite Phosphorylation.

Solid-organ transplant recipients exhibiting HLA donor-specific Abs are at risk for graft loss due to chronic Ab-mediated rejection. HLA Abs bind HLA molecules expressed on the surface of endothelial cells (ECs) and induce intracellular signaling pathways, including the activation of the transcriptional coactivator yes-associated protein (YAP). In this study, we examined the impact of lipid-lowering drugs of the statin family on YAP localization, multisite phosphorylation, and transcriptional activity in human ECs. Exposure of sparse cultures of ECs to cerivastatin or simvastatin induced striking relocalization of YAP from the nucleus to the cytoplasm and inhibited the expression of the YAP/TEA domain DNA-binding transcription factor-regulated genes connective tissue growth factor and cysteine-rich angiogenic inducer 61. In dense cultures of ECs, statins prevented YAP nuclear import and expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61 stimulated by the mAb W6/32 that binds HLA class I. Exposure of ECs to either cerivastatin or simvastatin completely blocked the migration of ECs stimulated by ligation of HLA class I. Exogenously supplied mevalonic acid or geranylgeraniol reversed the inhibitory effects of statins on YAP localization either in low-density ECs or high-density ECs challenged with W6/32. Mechanistically, cerivastatin increased the phosphorylation of YAP at Ser127, blunted the assembly of actin stress fiber, and inhibited YAP phosphorylation at Tyr357 in ECs. Using mutant YAP, we substantiated that YAP phosphorylation at Tyr357 is critical for YAP activation. Collectively, our results indicate that statins restrain YAP activity in EC models, thus providing a plausible mechanism underlying their beneficial effects in solid-organ transplant recipients.

Phosphorylation refines the control of complex formation in IDPs.

Dynamic and multivalent interactions are hallmarks of intrinsically disordered proteins (IDPs) acting within complex cellular regulatory pathways. Thanks to their astonishing molecular plasticity, IDPs often interact with numerous binding partners. While the interaction networks are expected to be highly regulated, disentangling the underlying regulatory mechanisms remains challenging. The nuclear coactivator binding domain (NCBD) is a known transcriptional hub in regulatory networks. At equilibrium, two conformations of NCBD exist due to the cis/trans isomerization of a single proline residue: an effect previously shown to expand the tunability of complex formation with the activator for thyroid hormone (ACTR) by orders of magnitude. Interestingly, proline residues are often located, as in the case of NCBD, within phosphorylation motifs.

Multilayered allosteric modulation of coupled folding and binding by phosphorylation, peptidyl-prolyl cis/trans isomerization, and diversity of interaction partners

Intrinsically disordered proteins (IDPs) play key roles in cellular regulation, including signal transduction, transcription, and cell-cycle control. Accordingly, IDPs can commonly interact with numerous different target proteins, and their interaction networks are expected to be highly regulated. However, many of the underlying regulatory mechanisms have remained unclear. Here, we examine the representative case of the nuclear coactivator binding domain (NCBD) of the large multidomain protein CBP, a hub in transcriptional regulation, and the interaction with several of its binding partners. Single-molecule Förster resonance energy transfer measurements show that phosphorylation of NCBD reduces its binding affinity, with effects that vary depending on the binding partner and the site and number of modifications. The complexity of the interaction is further increased by the dependence of the affinities on peptidyl-prolyl cis/trans isomerization in NCBD. Overall, our results reveal the potential for allosteric regulation on at least three levels: the different affinities of NCBD for its different binding partners, the differential modulation of these affinities by phosphorylation, and the effect of peptidyl-prolyl cis/trans isomerization on binding.

UBR5 Is a Hect E3 Ubiquitin Ligase That Regulates Chromatin Bound Nuclear Hormone Receptor Stability

Acute promyelocytic leukemia (APML) is driven by the oncogenic PML-RARA fusion, which is unique in that it is degraded by two small molecules, all-trans retinoic acid (ATRA) and arsenic trioxide. ATRA-induced degradation of retinoic acid receptor (RARA) is necessary for sustained anti-tumor effect (Ablain et al, JEM, 2013). More generally, nuclear hormone receptors (NRs) are degraded through a ligand-dependent manner of an unknown mechanism. Here we demonstrate that the HECT E3 ubiquitin ligase UBR5 is a ligand-dependent regulator of RARA and other NRs, solve its structure, and show it binds to NRs on chromatin, regulating transcription by degradation. We performed CRISPR-Cas9 screens on RARA and its partner, retinoid X receptor alpha (RXRA), to identify the ubiquitin ligase responsible for ATRA-dependent degradation. We found that UBR5 sgRNAs rescued ATRA-induced degradation. Moreover, we found that RARA and RXRA bind to UBR5 only in the presence of ATRA using co-immunoprecipitation experiments. NRs undergo a stereotypic structural change in the ligand binding domain (LBD) following ligand binding, revealing a conserved hydrophobic cleft that allows for nuclear co-activator (NCOA) binding through LxxLL peptide motifs, which are also present in UBR5. We performed mutagenesis screens on the RARA LBD and identified key residues within the cleft that, when altered, prevented ATRA-induced degradation and UBR5-RARA binding. Mutation of UBR5 LxxLL motifs also led to deficient RARA binding. We solved a 1.7Å crystal structure of RARA bound to a UBR5 LxxLL peptide, confirming ATRA dependent UBR5 binding in the cleft. To understand UBR5/RARA interactions in the context of full-length UBR5, we solved its structure via cryo-electron microscopy and performed a detailed characterization of its functional domains and binding sites. We confirmed the HECT domain is needed for degradation through a UBR5 CRISPR tiling screen and showed specific loss of function of the HECT domain through targeted sgRNAs led to a rescue of ATRA induced RARA degradation. RARA and PML-RARA are tightly chromatin associated, especially in the context of APML. To examine whether UBR5 binds RARA on chromatin, we performed chromatin-immunoprecipitation and sequencing (ChIP-seq) with anti-RARA and anti-UBR5 antibodies in an APML line (NB4) with and without ATRA treatment. Consistent with ATRA-induced degradation, we found significant loss of RARA peaks after ATRA treatment. RARA binding co-localized with UBR5 binding, suggesting UBR5 associates with RARA on chromatin. RNA-seq analysis of NB4 cells with or without UBR5 KD revealed significant alterations in transcript expression, implying that UBR5 regulates RARA-dependent transcription by degradation and therefore potentially downstream cellular behavior. To test this, we knocked down UBR5 in NB4s, treated with ATRA, and found that UBR5 KD led to increased ATRA resistance. More broadly, we found that UBR5 KD rescues ligand-induced degradation of other NRs including the glucocorticoid (GR), estrogen, vitamin-D, progesterone receptors and liver-X-receptor alpha. Through similar experiments we showed that UBR5 binds to GR in the presence of dexamethasone and recognizes and competes with NCOAs for a similar degron. ChIP and RNA-seq in A549 cells following dexamethasone treatment revealed that UBR5 also co-localizes with GR and regulates its transcription, suggesting UBR5-mediated degradation may be a general regulator of multiple NRs and their transcriptional activities. Our findings reveal a novel molecular mechanism regulating nuclear hormone receptors: ligand dependent E3 ligase binding on chromatin, leading to degradation and transcriptional regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Biochemical Identification of a Nuclear Coactivator Protein Required for AtrR-Dependent Gene Regulation in Aspergillus fumigatus.

Azole drugs represent the primary means of treating infections associated with the filamentous fungal pathogen Aspergillus fumigatus. A central player in azole resistance is the Zn2Cys6 zinc cluster-containing transcription factor AtrR. This factor stimulates expression of both the cyp51A gene, which encodes the azole drug target enzyme, as well as an ATP-binding cassette transporter-encoding gene called abcG1 (cdr1B). We used a fusion protein between AtrR and the tandem affinity purification (TAP) moiety to purify proteins that associated with AtrR from A. fumigatus. Protein fractions associated with AtrR-TAP were subjected to multidimensional protein identification technology mass spectrometry, and one of the proteins identified was encoded by the AFUA_6g08010 gene. We have designated this protein NcaA (for nuclear coactivator of AtrR). Loss of ncaA caused a reduction in voriconazole resistance and drug-induced abcG1 expression, although it did not impact induction of cyp51A transcription. We confirmed the association of AtrR and NcaA by coimmunoprecipitation from otherwise-wild-type cells. Expression of fusion proteins between AtrR and NcaA with green fluorescent protein allowed determination that these two proteins were localized in the A. fumigatus nucleus. Together, these data support the view that NcaA is required for nuclear gene transcription controlled by AtrR. IMPORTANCE Aspergillus fumigatus is a major filamentous fungal pathogen in humans and is susceptible to the azole antifungal class of drugs. However, loss of azole susceptibility has been detected with increasing frequency in the clinic, and infections associated with these azole-resistant isolates have been linked to treatment failure and worse outcomes. Many of these azole-resistant strains contain mutant alleles of the cyp51A gene, which encodes the azole drug target. A transcription factor essential for cyp51A gene transcription has been identified and designated AtrR. AtrR is required for azole-inducible cyp51A transcription, but we know little of the regulation of this transcription factor. Using a biochemical approach, we identified a new protein called NcaA that is involved in regulation of AtrR at certain target gene promoters. Understanding the mechanisms controlling AtrR function is an important goal in preventing or reversing azole resistance in this pathogen.

Cutting Edge: The Expression of Transcription Inhibitor GFI1 Is Induced by Retinoic Acid to Rein in Th9 Polarization.

IL-9, produced mainly by specialized T cells, mast cells, and group 2 innate lymphoid cells, regulates immune responses, including anti-helminth and allergic responses. Polarization of naive CD4 T cells into IL-9-producing T cells (Th9s) is induced by IL-4 and TGF-β1 or IL-1β. In this article, we report that the transcription factor growth factor-independent 1 transcriptional repressor (GFI1) plays a negative role in mouse Th9 polarization. Moreover, the expression of GFI1 is controlled by liganded RARα, allowing GFI1 to mediate the negative effect of retinoic acid on IL-9 expression. The Gfi1 gene has multiple RARα binding sites in the promoter region for recruiting nuclear coactivator steroid receptor coactivator-3 and p300 for histone epigenetic modifications in a retinoic acid-dependent manner. Retinoic acid-induced GFI1 binds the Il9 gene and suppresses its expression. Thus, GFI1 is a novel negative regulator of Il9 gene expression. The negative GFI1 pathway for IL-9 regulation provides a potential control point for Th9 activity.

Silencing the Adipocytokine NOV: A Novel Approach to Reversing Oxidative Stress-Induced Cardiometabolic Dysfunction.

Objective: NOV/CCN3 is an adipocytokine recently linked to obesity, insulin resistance, and cardiometabolic dysfunction. NOV is manufactured and secreted from adipose tissue, with blood levels highly correlated with BMI. NOV levels are increased in obesity and a myriad of inflammatory diseases. Elevated NOV levels cause oxidative stress by increasing free radicals, decreasing antioxidants, and decreasing heme oxygenase (HO-1) levels, resulting in decreased vascular function. Silencing NOV in NOV knockout mice improved insulin sensitivity. We wanted to study how suppressing NOV expression in an obese animal model affected pathways and processes related to obesity, inflammation, and cardiometabolic function. This is the first study to investigate the interaction of adipose tissue-specific NOV/CCN3 and cardiometabolic function. Methods: We constructed a lentivirus containing the adiponectin-promoter-driven shNOV to examine the effect of NOV inhibition (shNOV) in adipose tissue on the heart of mice fed a high-fat diet. Mice were randomly divided into three groups (five per group): (1) lean (normal diet), (2) high-fat diet (HFD)+ sham virus, and (3) HFD + shNOV lentivirus. Blood pressure, tissue inflammation, and oxygen consumption were measured. Metabolic and mitochondrial markers were studied in fat and heart tissues. Results: Mice fed an HFD developed adipocyte hypertrophy, fibrosis, inflammation, and decreased mitochondrial respiration. Inhibiting NOV expression in the adipose tissue of obese mice by shNOV increased mitochondrial markers for biogenesis (PGC-1α, the nuclear co-activator of HO-1) and functional integrity (FIS1) and insulin signaling (AKT). The upregulation of metabolic and mitochondrial markers was also evident in the hearts of the shNOV mice with the activation of mitophagy. Using RNA arrays, we identified a subgroup of genes that highly correlated with increased adipocyte mitochondrial autophagy in shNOV-treated mice. A heat map analysis in obese mice confirmed that the suppression of NOV overrides the genetic susceptibility of adiposity and the associated detrimental metabolic changes and correlates with the restoration of anti-inflammatory, thermogenic, and mitochondrial genes. Conclusion: Our novel findings demonstrate that inhibiting NOV expression improves adipose tissue function in a positive way in cardiometabolic function by inducing mitophagy and improving mitochondrial function by the upregulation of PGC-1α, the insulin sensitivity signaling protein. Inhibiting NOV expression increases PGC-1, a key component of cardiac bioenergetics, as well as key signaling components of metabolic change, resulting in improved glucose tolerance, improved mitochondrial function, and decreased inflammation. These metabolic changes resulted in increased oxygen consumption, decreased adipocyte size, and improved cardiac metabolism and vascular function at the structural level. The crosstalk of the adipose tissue-specific deletion of NOV/CCN3 improved cardiovascular function, representing a novel therapeutic strategy for obesity-related cardiometabolic dysfunction.

Mutant WDR45 Leads to Altered Ferritinophagy and Ferroptosis in β-Propeller Protein-Associated Neurodegeneration.

Beta-propeller protein-associated neurodegeneration (BPAN) is a subtype of neurodegeneration with brain iron accumulation (NBIA) caused by loss-of-function variants in WDR45. The underlying mechanism of iron accumulation in WDR45 deficiency remains elusive. We established a primary skin fibroblast culture of a new BPAN patient with a missense variant p.(Asn61Lys) in WDR45 (NM_007075.3: c.183C>A). The female patient has generalized dystonia, anarthria, parkinsonism, spasticity, stereotypies, and a distinctive cranial MRI with generalized brain atrophy, predominantly of the cerebellum. For the functional characterization of this variant and to provide a molecular link of WDR45 and iron accumulation, we looked for disease- and variant-related changes in the patient’s fibroblasts by qPCR, immunoblotting and immunofluorescence comparing to three controls and a previously reported WDR45 patient. We demonstrated molecular changes in mutant cells comprising an impaired mitochondrial network, decreased levels of lysosomal proteins and enzymes, and altered autophagy, confirming the pathogenicity of the variant. Compared to increased levels of the ferritinophagy marker Nuclear Coactivator 4 (NCOA4) in control cells upon iron treatment, patients’ cells revealed unchanged NCOA4 protein levels, indicating disturbed ferritinophagy. Additionally, we observed abnormal protein levels of markers of the iron-dependent cell death ferroptosis in patients’ cells. Altogether, our data suggests that WDR45 deficiency affects ferritinophagy and ferroptosis, consequentially disturbing iron recycling.

The clinicopathological and prognostic significances of LATS1 expression in breast cancer.

Large tumor suppressor gene 1 (LATS1) belongs to the PKA/PKG/PKC serine/threonine kinase subfamily of the Hippo signaling pathway and inactivates nuclear co-activators YAP1 and WWTR1 by phosphorylation. This study aimed to discern the clinicopathological and prognostic significances of LATS1 expression in breast cancer. We examined LATS1 expression in breast carcinogenesis and compared it with clinicopathological parameters and survival information of breast cancer patients using immunohistochemistry, western blotting, RT-PCR, and bioinformatics analysis. LATS1 expression was downregulated in breast cancer at both mRNA and protein levels (P<0.05). LATS1 mRNA expression was negatively correlated with low ER and PR expression, aggressive subtypes (TNBC and HER2+ vs. luminal), and poor survival (P<0.05). Its protein expression was negatively linked to patient age, T stage, N stage, M stage histological grade, PR status, and unfavorable prognosis (P<0.05). There was a positive correlationship between nuclar and cytoplasmic LATS1 expression in breast cancer (P<0.05). The downregulation of LATS1 expression plays a vital role in the carcinogenesis and progression of breast cancer. Thus, LATS1 loss was employed to indicate the aggressive behaviors and poor prognosis of breast cancer.

Nuclear receptor coactivator SRC-1 promotes colorectal cancer progression through enhancing GLI2-mediated Hedgehog signaling.

Overexpression of nuclear coactivator steroid receptor coactivator 1 (SRC-1) and aberrant activation of the Hedgehog (Hh) signaling pathway are associated with various tumorigenesis; however, the significance of SRC-1 in colorectal cancer (CRC) and its contribution to the activation of Hh signaling are unclear. Here, we identified a conserved Hh signaling signature positively correlated with SRC-1 expression in CRC based on TCGA database; SRC-1 deficiency significantly inhibited the proliferation, survival, migration, invasion, and tumorigenesis of both human and mouse CRC cells, and SRC-1 knockout significantly suppressed azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC in mice. Mechanistically, SRC-1 promoted the expression of GLI family zinc finger 2 (GLI2), a major downstream transcription factor of Hh pathway, and cooperated with GLI2 to enhance multiple Hh-regulated oncogene expression, including Cyclin D1, Bcl-2, and Slug. Pharmacological blockages of SRC-1 and Hh signaling retarded CRC progression in human CRC cell xenograft mouse model. Together, our studies uncover an SRC-1/GLI2-regulated Hh signaling looping axis that promotes CRC tumorigenesis, offering an attractive strategy for CRC treatment.

The dynamic properties of a nuclear coactivator binding domain are evolutionarily conserved

Evolution of proteins is constrained by their structure and function. While there is a consensus that the plasticity of intrinsically disordered proteins relaxes the structural constraints on evolution there is a paucity of data on the molecular details of these processes. The Nuclear Coactivator Binding Domain (NCBD) from CREB-binding protein is a protein interaction domain, which contains a hydrophobic core but is not behaving as a typical globular domain, and has been described as ‘molten-globule like’. The highly dynamic properties of NCBD makes it an interesting model system for evolutionary structure-function investigation of intrinsically disordered proteins. We have here compared the structure and biophysical properties of an ancient version of NCBD present in a bilaterian animal ancestor living around 600 million years ago with extant human NCBD. Using a combination of NMR spectroscopy, circular dichroism and kinetics we show that although NCBD has increased its thermodynamic stability, it has retained its dynamic biophysical properties in the ligand-free state in the evolutionary lineage leading from the last common bilaterian ancestor to humans. Our findings suggest that the dynamic properties of NCBD have been maintained by purifying selection and thus are important for its function, which includes mediating several distinct protein-protein interactions.