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Background: Prior research has highlighted the involvement of a transcriptional complex comprising C-terminal binding protein 2 (CtBP2), histone acetyltransferase p300, and nuclear factor kappa B (NF-κB) in the transactivation of proinflammatory cytokine genes, contributing to inflammation in mice with acute respiratory distress syndrome (ARDS). Nonetheless, it remains uncertain whether the therapeutic targeting of the CtBP2-p300-NF-κB complex holds potential for ARDS suppression. Methods: An ARDS mouse model was established using lipopolysaccharide (LPS) exposure. RNA-Sequencing (RNA-Seq) was performed on ARDS mice and LPS-treated cells with CtBP2, p300, and p65 knockdown. Small molecules inhibiting the CtBP2-p300 interaction were identified through AlphaScreen. Gene and protein expression levels were quantified using RT-qPCR and immunoblots. Tissue damage was assessed via histological staining. Key findings: We elucidated the specific role of the CtBP2-p300-NF-κB complex in proinflammatory gene regulation. RNA-seq analysis in LPS-challenged ARDS mice and LPS-treated CtBP2-knockdown (CtBP2KD), p300KD, and p65KD cells revealed its significant impact on proinflammatory genes with minimal effects on other NF-κB targets. Commercial inhibitors for CtBP2, p300, or NF-κB exhibited moderate cytotoxicity in vitro and in vivo, affecting both proinflammatory genes and other targets. We identified a potent inhibitor, PNSC928, for the CtBP2-p300 interaction using AlphaScreen. PNSC928 treatment hindered the assembly of the CtBP2-p300-NF-κB complex, substantially downregulating proinflammatory cytokine gene expression without observable cytotoxicity in normal cells. In vivo administration of PNSC928 significantly reduced CtBP2-driven proinflammatory gene expression in ARDS mice, alleviating inflammation and lung injury, ultimately improving ARDS prognosis. Conclusion: Our results position PNSC928 as a promising therapeutic candidate to specifically target the CtBP2-p300 interaction and mitigate inflammation in ARDS management. [ABSTRACT FROM AUTHOR]

C-terminal binding protein (CtBP), a transcriptional co-repressor, significantly influences cellular signaling, impacting various biological processes including cell proliferation, differentiation, apoptosis, and immune responses. The CtBP family comprises two highly conserved proteins, CtBP1 and CtBP2, which have been shown to play critical roles in both tumorigenesis and the regulation of viral infections. Elevated CtBP expression is noted in various tumor tissues, promoting tumorigenesis, invasiveness, and metastasis through multiple pathways. Additionally, CtBP's role in viral infections varies, exhibiting differing or even opposing effects depending on the virus. This review synthesizes the advances in CtBP's function research in viral infections and virus-associated tumorigenesis, offering new insights into potential antiviral and anticancer strategies. [ABSTRACT FROM AUTHOR]

Aims The activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in endothelial cells (ECs) contributes to vascular inflammation in atherosclerosis. Considering the high glycolytic rate of ECs, we delineated whether and how glycolysis determines endothelial NLRP3 inflammasome activation in atherosclerosis. Methods and results Our results demonstrated a significant up-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key regulator of glycolysis, in human and mouse atherosclerotic endothelium, which positively correlated with NLRP3 levels. Atherosclerotic stimuli up-regulated endothelial PFKFB3 expression via sterol regulatory element-binding protein 2 (SREBP2) transactivation. EC-selective haplodeficiency of Pfkfb3 in Apoe−/− mice resulted in reduced endothelial NLRP3 inflammasome activation and attenuation of atherogenesis. Mechanistic investigations revealed that PFKFB3-driven glycolysis increased the NADH content and induced oligomerization of C-terminal binding protein 1 (CtBP1), an NADH-sensitive transcriptional co-repressor. The monomer form, but not the oligomer form, of CtBP1 was found to associate with the transcriptional repressor Forkhead box P1 (FOXP1) and acted as a transrepressor of inflammasome components, including NLRP3, caspase-1, and interleukin-1β (IL-1β). Interfering with NADH-induced CtBP1 oligomerization restored its binding to FOXP1 and inhibited the glycolysis-dependent up-regulation of NLRP3, Caspase-1, and IL-1β. Additionally, EC-specific overexpression of NADH-insensitive CtBP1 alleviates atherosclerosis. Conclusion Our findings highlight the existence of a glycolysis-dependent NADH/CtBP/FOXP1-transrepression pathway that regulates endothelial NLRP3 inflammasome activation in atherogenesis. This pathway represents a potential target for selective PFKFB3 inhibitors or strategies aimed at disrupting CtBP1 oligomerization to modulate atherosclerosis. [ABSTRACT FROM AUTHOR]

Background: Prostate cancer is dependent on androgen receptor (AR) signaling, and androgen deprivation therapy (ADT) has proven effective in targeting prostate cancer. However, castration-resistant prostate cancer (CRPC) eventually emerges. AR signaling inhibitors (ARSI) have been also used, but resistance to these agents develops due to genetic AR alterations and epigenetic dysregulation. Methods: In this study, we investigated the role of OCT1, a member of the OCT family, in an AR-positive CRPC patient-derived xenograft established from a patient with resistance to ARSI and chemotherapy. We conducted a genome-wide analysis chromatin immunoprecipitation followed by sequencing and bioinformatic analyses using public database. Results: Genome-wide analysis of OCT1 target genes in PDX 201.1 A revealed distinct OCT1 binding sites compared to treatment-naïve cells. Bioinformatic analyses revealed that OCT1-regulated genes were associated with cell migration and immune system regulation. In particular, C-terminal Binding Protein 2 (CTBP2), an OCT1/AR target gene, was correlated with poor prognosis and immunosuppressive effects in the tumor microenvironment. Metascape revealed that CTBP2 knockdown affects genes related to the immune response to bacteria. Furthermore, TISIDB analysis suggested the relationship between CTBP2 expression and immune cell infiltration in prostate cancer, suggesting that it may contribute to immune evasion in CRPC. Conclusions: Our findings shed light on the genome-wide network of OCT1 and AR in AR-positive CRPC and highlight the potential role of CTBP2 in immune response and tumor progression. Targeting CTBP2 may represent a promising therapeutic approach for aggressive AR-positive CRPC. Further validation will be required to explore novel therapeutic strategies for CRPC management. [ABSTRACT FROM AUTHOR]

EH domain‐containing protein 2 (EHD2) is a member of the EHD protein family and is mainly located in the plasma membrane, but can also be found in the cytoplasm and endosomes. EHD2 is also a nuclear‐cytoplasmic shuttle protein. After entering the cell nuclear, EHD2 acts as a corepressor of transcription to inhibit gene transcription. EHD2 regulates a series of biological processes. As a key regulator of endocytic transport, EHD2 is involved in the formation and maintenance of endosomal tubules and vesicles, which are critical for the intracellular transport of proteins and other substances. The N‐terminal of EHD2 is attached to the cell membrane, while its C‐terminal binds to the actin‐binding protein. After binding, EHD2 connects with the actin cytoskeleton, forming the curvature of the membrane and promoting cell endocytosis. EHD2 is also associated with membrane protein trafficking and receptor signaling, as well as in glucose metabolism and lipid metabolism. In this review, we highlight the recent advances in the function of EHD2 in various cellular processes and its potential implications in human diseases such as cancer and metabolic disease. We also discussed the prospects for the future of EHD2. EHD2 has a broad prospect as a therapeutic target for a variety of diseases. Further research is needed to explore its mechanism, which could pave the way for the development of targeted treatments. Significance statement: The overview and biological function of EH domain‐containing protein 2 (EHD2) are discussed in this article.The article focuses on the role of EHD2 in metabolic diseases and cancer.The article also discussed the prospects for the future of EHD2 (Figure 1). [ABSTRACT FROM AUTHOR]

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor molecule involved in several cellular signaling cascades. Here, we attempted to identify novel STAP-2 interacting molecules, and identified c-Cbl associated protein (CAP) as a binding protein through the C-terminal proline-rich region of STAP-2. Expression of STAP-2 increased the interaction between CAP and c-Cbl, suggesting that STAP-2 bridges these proteins and enhances complex formation. CAP/c-Cbl complex is known to regulate GLUT4 translocation in insulin signaling. STAP-2 overexpressed human hepatocyte Hep3B cells showed enhanced GLUT4 translocation after insulin treatment. Elevated levels of Stap2 mRNA have been observed in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs) during adipocyte differentiation. The differentiation of 3T3-L1 cells into adipocytes was highly promoted by retroviral overexpression of STAP-2. In contrast, STAP-2 knockout (KO) MEFs exhibited suppressed adipogenesis. The increase in body weight with high-fat diet feeding was significantly decreased in STAP-2 KO mice compared to WT animals. These data suggest that the expression of STAP-2 correlates with adipogenesis. Thus, STAP-2 is a novel regulatory molecule that controls insulin signal transduction by forming a c-Cbl/STAP-2/CAP ternary complex. [ABSTRACT FROM AUTHOR]

Hypotonia, Ataxia, Developmental Delay, and Tooth Enamel Defect Syndrome (HADDTS) is an exceptionally rare disorder resulting from a heterozygous variant in the C-terminal binding protein 1 (CTBP1) gene. To date, a mere two variants (14 patients) have been documented on a global scale. The aim of this study was to identify a causative CTBP1 variant in a Chinese patient, and to determine the potential pathogenicity of the identified variant. Here, Wholeexome sequencing (WES) was conducted on the proband to pinpoint the candidate variant. Following this, Sanger sequencing was employed to validate the identified candidate variant and examine its co-segregation within the available family members. Employing both in silico prediction and three-dimensional protein modeling, we conducted an analysis to assess the potential functional implications of the variant on the encoded protein. Our investigation led to the identification of a novel heterozygous variant in the CTBP1 gene, namely, c.371 C>T (p.Ser124Phe), in a Chinese patient. This case represents the first confirmed instance of such a variant in a Chinese patient. When comparing the patient's clinical symptoms with those reported in the literature, notable distinctions were observed between her primary symptoms and those associated with HADDTS. She showed other signs such as microcephaly, coarse facial features, single transverse palmar crease, visible beard, myopia, coarse toenail and skeletal anomalies. This study enriching the spectrum of genetic variants observed in different ethnic populations and expanding the phenotypic profile associated with this gene. These findings are expected to contribute to the enhancement of future variant-based screening and genetic diagnosis, while also providing further insights into the pathogenic mechanisms underlying CTBP1-related conditions. [ABSTRACT FROM AUTHOR]

Innovative therapeutic strategies for esophageal squamous cell carcinoma (ESCC) are urgently required due to the limited effectiveness of standard chemotherapies. C-Terminal Binding Protein 1 (CtBP1) has been implicated in various cancers, including ESCC. However, the precise expression patterns and functional roles of CtBP1 in ESCC remain inadequately characterized. In this study, we aimed to investigate CtBP1 expression and its role in the resistance of ESCC to paclitaxel, an effective chemotherapeutic agent. Western blotting and immunofluorescence were applied to assess CtBP1 expression in the TE-1 and KYSE-50 cell lines. We observed the marked expression of CtBP1, which was associated with enhanced proliferation, invasion, and metastasis in these cell lines. Further, we successfully generated paclitaxel resistant ESCC cell lines and conducted cell viability assays. We employed the CRISPR/Cas9 genome editing system to disable the CtBP1 gene in ESCC cell lines. Through the analysis of the drug dose–response curve, we assessed the sensitivity of these cell lines in different treatment groups. Remarkably, CtBP1-disabled cell lines displayed not only improved sensitivity but also a remarkable inhibition of proliferation, invasion, and metastasis. This demonstrates that CtBP1 may promote ESCC cell malignancy and confer paclitaxel resistance. In summary, our study opens a promising avenue for targeted therapies, revealing the potential of CtBP1 inhibition to enhance the effectiveness of paclitaxel treatment for the personalized management of ESCC. [ABSTRACT FROM AUTHOR]

C-terminal binding proteins (CtBP) are transcriptional co-repressors regulating gene expression. CtBP promote neuronal survival through repression of pro-apoptotic genes, and may represent relevant targets for neurodegenerative disorders, such as Parkinson's disease (PD). Nevertheless, evidence of the role of CtBP1 and CtBP2 in neurodegeneration are scarce. Herein, we showed that CtBP1 and CtBP2 are expressed in neurons, dopaminergic neurons, astrocytes, and microglia in the substantia nigra (SN) and striatum of adult mice. Old mice showed a lower expression of CtBP1 in the SN and higher expression of CtPB2 in the SN and striatum compared with adult mice. In vivo models for PD (paraquat, MPTP, 6-OHDA) showed increased expression of CtBP1 in the SN and striatum while CtBP2 expression was increased in the striatum of paraquat-treated rats only. Moreover, an increased expression of both CtBP was found in a dopaminergic cell line (N27) exposed to 6-OHDA. In the 6-OHDA PD model, we found a dual effect using an unspecific ligand of CtBP, the 4-methylthio 2-oxobutyric acid (MTOB): higher concentrations (e.g. 2500 µM, 1000 µM) inhibited dopaminergic survival, while at 250 μM it counteracted cell death. In vitro, this latter protective role was absent after the siRNA silencing of CtBP1 or CtBP2. Altogether, this is the first report exploring the cellular and regional expression pattern of CtBP in the nigrostriatal pathway and the neuroprotective role in PD toxin-based models. CtBP could counteract dopaminergic cell death in the 6-OHDA PD model and, therefore, CtBP function and therapeutic potential in PD should be further explored. [ABSTRACT FROM AUTHOR]

Cell adhesion molecules, including integrins, cadherins, and claudins (CLDNs), are known to activate Src-family kinases (SFKs) that organize a variety of physiological and pathological processes; however, the underlying molecular basis remains unclear. Here, we identify the SFK members that are coupled with the CLDN6-adhesion signaling. Among SFK subtypes, BLK, FGR, HCK, and SRC were highly expressed in F9 cells and concentrated with CLDN6 along cell borders during epithelial differentiation. Immunoprecipitation assay showed that BLK and SRC, but not FGR or HCK, form a complex with CLDN6 via the C-terminal cytoplasmic domain. We also demonstrated, by pull-down assay, that recombinant BLK and SRC proteins directly bind to the C-terminal cytoplasmic domain of CLDN6 (CLDN6C). Unexpectedly, both recombinant SFK proteins recognized the CLDN6C peptide in a phosphotyrosine-independent manner. Furthermore, by comparing phenotypes of F9:Cldn6:Blk−/− and F9:Cldn6:Src−/− cells with those of wild-type F9 and F9:Cldn6 cells, we revealed that BLK and SRC are essential for CLDN6-triggered cellular events, namely epithelial differentiation and the expression of retinoid acid receptor target genes. These results indicate that selective SFK members appear to participate in the CLDN-adhesion signaling. [ABSTRACT FROM AUTHOR]

Introduction: Positive regulatory domain containing 16 (PRDM16) protein represents the key regulator of brown adipose tissue (BAT) development. It induces brown fat phenotype and represses white adipose tissue specific genes through the association with C-terminal binding co-repressor proteins (CtBP1 and CtBP2). In healthy adults presence of BAT has been associated with lower glucose, total cholesterol and low-density lipoprotein (LDL) cholesterol levels. Our aim was to analyze the association of PRDM16 gene (rs12409277) and CtBP2 gene (rs1561589) polymorphisms with body mass index (BMI), fasting glucose level and lipid profile of adolescents. Material and methods: Our study included 295 healthy school children, 145 boys (49.2%) and 150 girls (50.8%), 15 years of age. Genotypes for the selected polymorphisms were detected by the real-time PCR method. Age, gender, height, weight, lipid profile (total cholesterol, high-density lipoprotein (HDL) cholesterol, LDL cholesterol, triglycerides) and fasting glucose levels were recorded. Results: We did not find a statistically significant association of rs12409277 and rs1561589 polymorphisms with BMI, fasting glucose and lipid profile of adolescents. We further analyzed the combined effect of the two SNPs and the statistical analysis showed that carriers of CT genotype of rs12409277 polymorphism and GG genotype of rs1561589 polymorphism had significantly lower total cholesterol (p = 0.001) and LDL cholesterol (p = 0.008) levels compared to all other groups of genotypes. Conclusions: Our study suggests that rs12409277 and rs1561589 polymorphism might have an influence on total and LDL cholesterol levels in adolescents. Larger studies should be performed in order to confirm our results. [ABSTRACT FROM AUTHOR]

Nuclear factor-κB (NF-κB)–mediated inflammation is a major cause of acute respiratory distress syndrome (ARDS). However, the regulatory mechanisms by which NF-κB transactivates proinflammatory cytokines remain unclear in the pathogenesis of ARDS. Herein, we report that the activating protein 1 (AP1) transcription factor recruits a histone acetyltransferase p300 and a transcriptional regulator C-terminal binding protein 1 (CtBP1) to assemble the CtBP1-p300-AP1 complex, which transactivates the expression of hsa-miR-7-5p in ARDS biopsies. Overexpressed hsa-miR-7-5p binds to the three prime untranslated regions (3′-UTRs) of ataxin 1 (ATXN1), suppressing its expression. Decreased ATXN1 expression relieves its repression of NF-κB, causing the induction of proinflammatory cytokine genes and triggering an inflammatory response. Depletion of CtBP1 or treatments with two CtBP1 inhibitors (NSC95397 and 4-methylthio-2-oxobutanoate (MTOB)) in human macrophages impairs the assembly of the CtBP2-p300-AP1 complex, resulting in decreased hsa-miR-7-5p levels, upregulation of ATXN1, and attenuation of proinflammatory cytokines. A similar regulatory mechanism was observed in lipopolysaccharide-treated mice. Our results reveal that increased hsa-miR-7-5p level mediated by the CtBP1-p300-AP1 complex targets ATXN1 to trigger an NF-κB-dependent inflammatory response. Interfering with this signaling pathway to block the inflammatory response may be a strategy for treating ARDS. Key messages: The transcription factor AP1 recruits p300 and CtBP1 to form a transcriptional complex, which transactivates the expression of hsa-miR-7-5p in ARDS biopsies. Overexpressed hsa-miR-7-5p binds to the 3′-UTR of ATXN1, suppressing its expression. The decreased ATXN1 impaired its suppression of NF-κB, causing the induction of proinflammatory cytokine genes and triggering inflammation response. Disruption of the assembly of CtBP2-p300-AP1 complex upregulates ATXN1 and attenuates inflammation. [ABSTRACT FROM AUTHOR]

Super-enhancers (SEs) regulate gene expressions, which are critical for cell type-identity and tumorigenesis. Although genome wide H3K27ac profiling have revealed the presence of SE-associated genes in gastric cancer (GC), their roles remain unclear. In this study, ChIP-seq and HiChIP-seq experiments revealed mitogen-activated protein kinase 8 (MAP3K8) to be an SE-associated gene with chromosome interactions in Epstein–Barr virus-associated gastric carcinoma (EBVaGC) cells. CRISPRi mediated repression of the MAP3K8 SEs attenuated MAP3K8 expression and EBVaGC cell proliferation. The results were validated by treating EBVaGC cells with bromodomain and the extra-terminal motif (BET) inhibitor, OTX015. Further, functional analysis of MAP3K8 in EBVaGC revealed that silencing MAP3K8 could inhibit the cell proliferation, colony formation, and migration of EBVaGC cells. RNA-seq and pathway analysis indicated that knocking down MAP3K8 obstructed the notch signaling pathway and epithelial-mesenchymal transition (EMT) in EBVaGC cells. Further, analysis of the cancer genome atlas (TCGA) and GSE51575 databases exhibited augmented MAP3K8 expression in gastric cancer and it was found to be inversely correlated with the disease-free progression of GC. Moreover, Spearman's correlation revealed that MAP3K8 expression was positively correlated with the expressions of notch pathway and EMT related genes, such as, Notch1, Notch2, C-terminal binding protein 2 (CTBP2), alpha smooth muscle actin isotype 2 (ACTA2), transforming growth factor beta receptor 1 (TGFβR1), and snail family transcriptional repressors 1/2 (SNAI1/SNAI2) in GC. Taken together, we are the first to functionally interrogate the mechanism of SE-mediated regulation of MAP3K8 in EBVaGC cell lines. [ABSTRACT FROM AUTHOR]

Evolution of sequence-specific transcription factors clearly drives lineage-specific innovations, but less is known about how changes in the central transcriptional machinery may contribute to evolutionary transformations. In particular, transcriptional regulators are rich in intrinsically disordered regions that appear to be magnets for evolutionary innovation. The C-terminal Binding Protein (CtBP) is a transcriptional corepressor derived from an ancestral lineage of alpha hydroxyacid dehydrogenases; it is found in mammals and invertebrates, and features a core NAD-binding domain as well as an unstructured C-terminus (CTD) of unknown function. CtBP can act on promoters and enhancers to repress transcription through chromatin-linked mechanisms. Our comparative phylogenetic study shows that CtBP is a bilaterian innovation whose CTD of about 100 residues is present in almost all orthologs. CtBP CTDs contain conserved blocks of residues and retain a predicted disordered property, despite having variations in the primary sequence. Interestingly, the structure of the C-terminus has undergone radical transformation independently in certain lineages including flatworms and nematodes. Also contributing to CTD diversity is the production of myriad alternative RNA splicing products, including the production of "short" tailless forms of CtBP in Drosophila. Additional diversity stems from multiple gene duplications in vertebrates, where up to five CtBP orthologs have been observed. Vertebrate lineages show fewer major modifications in the unstructured CTD, possibly because gene regulatory constraints of the vertebrate body plan place specific constraints on this domain. Our study highlights the rich regulatory potential of this previously unstudied domain of a central transcriptional regulator. [ABSTRACT FROM AUTHOR]

Aminoglycosides exhibit ototoxicity by damaging mitochondria, which in turn generate reactive oxygen species that induce hair cell death and subsequent hearing loss. It is well known that damaged mitochondria are degraded by mitophagy, an important mitochondrial quality control system that maintains mitochondrial homeostasis and ensures cell survival. However, it is unclear whether dysregulation of mitophagy contributes to aminoglycoside-induced hair cell injury. In the current study, we found that PINK1-PRKN-mediated mitophagy was impaired in neomycin-treated hair cells. Our data suggested that mitochondrial recruitment of PRKN and phagophore recognition of damaged mitochondria during mitophagy were blocked following neomycin treatment. In addition, the degradation of damaged mitochondria by lysosomes was significantly decreased as indicated by the mitophagic flux reporter mt-mKeima. Moreover, we demonstrated that neomycin disrupted mitophagy through transcriptional inhibition of Pink1 expression, the key initiator of mitophagy. Moreover, we found that neomycin impaired mitophagy by inducing ATF3 expression. Importantly, treatment with a mitophagy activator could rescue neomycin-treated hair cells by increasing mitophagy, indicating that genetic modulation or drug intervention in mitophagy may have therapeutic potential for aminoglycoside-induced hearing loss. Abbreviations: AAV: adeno-associated virus; ABR: auditory brainstem response; ATF3: activating transcription factor 3; ATOH1/MATH1: atonal bHLH transcription factor 1; BafA1: bafilomycin A1; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; COX4I1/COXIV: cytochrome c oxidase subunit 4I1; CTBP2/RIBEYE: C-terminal binding protein 2; DFP: deferiprone; EGFP: enhanced green fluorescent protein; FOXO3: forkhead box O3; GRIA2/GLUR2: glutamate receptor, ionotropic, AMPA2 (alpha 2); HC: hair cell; HSPD1/HSP60: heat shock protein 1 (chaperonin); IHC: inner hair cell; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MYO7A: myosin VIIA; OPTN: optineurin; OMM: outer mitochondrial membrane; PRKN: parkin RBR E3 ubiquitin protein ligase; PINK1: PTEN induced putative kinase 1; RT-qPCR: real-time quantitative polymerase chain reaction; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; TUNEL: Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling; USP30: ubiquitin specific peptidase 30; XBP1: X-box binding protein 1. [ABSTRACT FROM AUTHOR]

The hippocampus participates in spatial navigation and behavioral processes, displays molecular plasticity in response to environmental challenges, and can play a role in neuropsychiatric diseases. The combined effects of inflammatory prenatal and postnatal challenges can disrupt the hippocampal gene networks and regulatory mechanisms. Using a proven pig model of viral maternal immune activation (MIA) matched to controls and an RNA-sequencing approach, the hippocampal transcriptome was profiled on two-month-old female and male offspring assigned to fasting, mimetic viral, or saline treatments. More than 2600 genes presented single or combined effects (FDR-adjusted p-value < 0.05) of MIA, postnatal stress, or sex. Biological processes and pathways encompassing messenger cyclic adenosine 3′,5′-monophosphate (cAMP) signaling were enriched with genes including gastric inhibitory polypeptide receptor (GIPR) predominantly over-expressed in the MIA-exposed fasting males relative to groups that differed in sex, prenatal or postnatal challenge. While this pattern was amplified in fasting offspring, the postnatal inflammatory challenge appeared to cancel out the effects of the prenatal challenge. The transcription factors C-terminal binding protein 2 (CTBP2), RE1 silencing transcription factor (REST), signal transducer and activator of transcription 1 (STAT1), and SUZ12 polycomb repressive complex 2 subunit were over-represented among the genes impacted by the prenatal and postnatal factors studied. Our results indicate that one environmental challenge can influence the effect of another challenge on the hippocampal transcriptome. These findings can assist in the identification of molecular targets to ameliorate the effects of pre-and post-natal stressors on hippocampal-associated physiology and behavior. [ABSTRACT FROM AUTHOR]

The nucleocapsid (N) protein plays critical roles in coronavirus genome transcription and packaging, representing a key target for the development of novel antivirals, and for which structural information on ligand binding is scarce. We used a novel fluorescence polarization assay to identify small molecules that disrupt the binding of the N protein to a target RNA derived from the SARS-CoV-2 genome packaging signal. Several phenolic compounds, including L-chicoric acid (CA), were identified as high-affinity N-protein ligands. The binding of CA to the N protein was confirmed by isothermal titration calorimetry, 1H-STD and 15N-HSQC NMR, and by the crystal structure of CA bound to the N protein C-terminal domain (CTD), further revealing a new modulatory site in the SARS-CoV-2 N protein. Moreover, CA reduced SARS-CoV-2 replication in cell cultures. These data thus open venues for the development of new antivirals targeting the N protein, an essential and yet underexplored coronavirus target. [ABSTRACT FROM AUTHOR]

C-terminal binding proteins (CtBP1/2) are transcriptional coregulators that play a significant role during vertebrate neurodevelopment. This systematic review aims to identify case reports with genetic variants in CTBP1 and CTBP2 associated with brain development syndromes. We screened different databases (PubMed, Scopus, Google Scholar, LILACS) by systematically searching journals and checking reference lists and citations of background papers. We found fourteen cases (10 males) from five papers carrying two pathogenic, heterozygous variants in the CTBP1 gene (13 individuals carried the missense mutation c.991C T, p.Arg342Trp, and one subject carrying the 2-base pair deletion c.1315_1316delCA, p.Gln439ValfsTer84). These mutations were de novo in 13 cases and one case of maternal germinal mosaicism. Two variants are in the same domain of the protein: Pro-Leu-Asp-Leu-Ser (PLDLS) C terminal. Patients with these mutations exhibit a phenotype with intellectual disability, HADDTS syndrome (hypotonia, ataxia, developmental delay, and tooth enamel defects), and cerebellar volume loss. We did not identify reported cases associated with homozygous mutations harbored in CTBP1. We did not identify any report of neurodevelopment phenotypes associated with heterozygous or homozygous CTBP2 mutations. Due to CTBP2/RIBEYE being a gene with dual function, identifying and interpreting the potential pathogenic variants is challenging. Further, homozygous mutations in the CTBP2 gene may be lethal. The mechanisms involved in the pathogenesis of neurodevelopment due to variants of these proteins have not yet been elucidated, despite some functional evidence. Further studies should be conducted to understand these transcription factors and their interaction with each other and their partners. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved C-terminal binding protein (CtBP) has been well characterized as a transcriptional co-repressor. Herein, we report a previously unreported function for CtBP, showing that lowering CtBP dosage genetically suppresses Polycomb group (PcG) loss-of-function phenotypes while enhancing that of trithorax group (trxG) in Drosophila, suggesting that the role of CtBP in gene activation is more pronounced in fly development than previously thought. In fly cells, we show that CtBP is required for the derepression of the most direct PcG target genes, which are highly enriched by homeobox transcription factors, including Hox genes. Using ChIP and co-IP assays, we demonstrate that CtBP is directly required for the molecular switch between H3K27me3 and H3K27ac in the derepressed Hox loci. In addition, CtBP physically interacts with many proteins, such as UTX, CBP, Fs(1)h and RNA Pol II, that have activation roles, potentially assisting in their recruitment to promoters and Polycomb response elements that control Hox gene expression. Therefore, we reveal a prominent activation function for CtBP that confers a major role for the epigenetic program of fly segmentation and development. [ABSTRACT FROM AUTHOR]

The overexpansion of adipose tissues leads to obesity and eventually results in metabolic disorders. Garcinia cambogia (G. cambogia) has been used as an antiobesity supplement. However, the molecular mechanisms underlying the effects of G. cambogia on cellular processes have yet to be fully understood. Here, we discovered that G. cambogia attenuated the expression of CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), an important adipogenic factor, suppressing its transcription in differentiated cells. In addition, G. cambogia inhibited macroautophagic/autophagic flux by decreasing autophagy-related gene expression and autophagosome formation. Notably, G. cambogia markedly elevated the expression of KLF3 (Kruppel-like factor 3 (basic)), a negative regulator of adipogenesis, by reducing SQSTM1/p62-mediated selective autophagic degradation. Furthermore, increased KLF3 induced by G. cambogia interacted with CTBP2 (C-terminal binding protein 2) to form a transcriptional repressor complex and inhibited Cebpa and Pparg transcription. Importantly, we found that RPS6KA1 and STAT3 were involved in the G. cambogia-mediated regulation of CEBPB and autophagic flux. In an obese animal model, G. cambogia reduced high-fat diet (HFD)-induced obesity by suppressing epididymal and inguinal subcutaneous white adipose tissue mass and adipocyte size, which were attributed to the regulation of targets that had been consistently identified in vitro. These findings provide new insight into the mechanism of G. cambogia-mediated regulation of adipogenesis and suggest molecular links to therapeutic targets for the treatment of obesity. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; ATG: autophagy-related; Baf: bafilomycin A1; BECN1: beclin 1; CEBP: CCAAT/enhancer binding protein (C/EBP); CHX: cycloheximide; CREB: cAMP response element binding protein; CTBP: C-terminal binding protein; EGCG: (-)-epigallocatechin gallate; eWAT: epididymal white; G. cambogia: Garcinia cambogia; GFP: green fluorescent protein; H&E: hematoxylin and eosin; HFD: high-fat diet; iWAT: inguinal subcutaneous white; KLF: Kruppel-like factor; LAP: liver-enriched transcriptional activating proteins; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; ND: normal diet; PPARG: peroxisome proliferator activated receptor gamma; qPCR: quantitative real-time PCR; RFP: red fluorescent protein; RPS6KA1: ribosomal protein S6 kinase A1; siRNA: small-interfering RNA; SQSTM1/p62: sequestosome 1; STAT: signal transducer and activator of transcription; TEM: transmission electron microscopy [ABSTRACT FROM AUTHOR]

Objective: Aberrant activation of Wnt/β-catenin signaling contributes to the maintenance of cancer stem cells and chemoresistance in colorectal cancer (CRC). Retinoic acid-induced 2 (RAI2) was proved to be a tumor suppressor in CRC in our previous report. In this study, the role of RAI2 in Wnt/β-catenin signaling was further investigated. Methods: As a transcriptional co-regulator, C-terminal Binding Protein 2 (CtBP2) was reported to be involved in Wnt signaling in multiple and complex ways. The correlation of RAI2 and CtBP2 in CRC was analyzed by TCGA dataset, and the interaction between RAI2 and CtBP2 was explored by co-immunoprecipitation (Co-IP) in CRC cells. The effect of RAI2 on the activity of Wnt signaling and the location of β-catenin was detected by Dual-Luciferase reporter assay and Immunofluorescence respectively. Western blotting analysis was performed to detect the expression of target genes involved in Wnt signaling. Sphere formation assay was employed to detect the effect of RAI2 on stem cell like properties. Cell viability assay was used to detect the chemosensitivity of cells before and after transfection of RAI2. Results: The interaction between RAI2 and CtBP2 was confirmed by Co-IP in CRC cells. Besides, the negative correlation of RAI2 and CtBP2 in CRC was found by analyzing the TCGA dataset. Re-expression of RAI2 in human colon cancer cells (HCT116 and LoVo) suppressed the fluorescent activity of Wnt signaling, increased the phosphorylation and inhibited nuclear translocation of β-catenin, with down-regulation of target genes like c-Myc, CyclinD1, ASCL2, and LGR5. In contrast, the mutated RAI2, which can't interact with CtBP2, has no above effects. We observed low expression of RAI2 in 33.89% (101/298) of CRC patients, which was significantly associated with reduced phosphorylation of β-catenin (r=0.8866, P<0.0001), poor 5-year relapse-free survival (RFS) (P = 0.0029) and overall survival (OS) (P = 0.0102). Restoration of RAI2 in HCT116 and LoVo cells inhibited stem cell-like properties of CRC cells and increased chemosensitivity of these cells to oxaliplatin and fluorouracil. Conclusion: Low expression of RAI2 can serve as an independent poor prognostic marker. RAI2 inhibits Wnt signaling by interacting with or down-regulating CtBP2, resulting in repression of stem cell-like properties and increased chemosensitivity of CRC cells. [ABSTRACT FROM AUTHOR]

The activation of oncogenic C‐terminal binding Protein (CtBP) transcriptional activity is coupled with NAD(H) binding and homo‐oligomeric assembly, although the level of CtBP assembly and nucleotide binding affinity continues to be debated. Here, we apply biophysical techniques to address these fundamental issues for CtBP1 and CtBP2. Our ultracentrifugation results unambiguously demonstrate that CtBP assembles into tetramers in the presence of saturating NAD+ or NADH with tetramer to dimer dissociation constants about 100 nm. Isothermal titration calorimetry measurements of NAD(H) binding to CtBP show dissociation constants between 30 and 500 nm, depending on the nucleotide and paralog. Given cellular levels of NAD+, CtBP is likely to be fully saturated with NAD under physiological concentrations suggesting that CtBP is unable to act as a sensor for NADH levels. [ABSTRACT FROM AUTHOR]

Biological systems to sense and respond to metabolic perturbations are critical for the maintenance of cellular homeostasis. Here we describe a hepatic system in this context orchestrated by the transcriptional corepressor C-terminal binding protein 2 (CtBP2) that harbors metabolite-sensing capabilities. The repressor activity of CtBP2 is reciprocally regulated by NADH and acyl-CoAs. CtBP2 represses Forkhead box O1 (FoxO1)-mediated hepatic gluconeogenesis directly as well as Sterol Regulatory Element-Binding Protein 1 (SREBP1)-mediated lipogenesis indirectly. The activity of CtBP2 is markedly defective in obese liver reflecting the metabolic perturbations. Thus, liver-specific CtBP2 deletion promotes hepatic gluconeogenesis and accelerates the progression of steatohepatitis. Conversely, activation of CtBP2 ameliorates diabetes and hepatic steatosis in obesity. The structure-function relationships revealed in this study identify a critical structural domain called Rossmann fold, a metabolite-sensing pocket, that is susceptible to metabolic liabilities and potentially targetable for developing therapeutic approaches. Sensing of nutrient status coordinates the regulation of liver glucose and lipid metabolism, and is important for metabolic homeostasis. Here the authors report that transcriptional the corepressor CtBP2 can sense nutrient status and coordinate repression of liver glucose and lipid metabolism via Fox01 and SREBP1, respectively. [ABSTRACT FROM AUTHOR]

Background: The zinc-finger transcription factor Krüppel-homolog 1 (Kr-h1) exerts a dual regulatory role during insect development by preventing precocious larval/nymphal metamorphosis and in stimulating aspects of adult reproduction such as vitellogenesis. However, how Kr-h1 functions both as a transcriptional repressor in juvenile metamorphosis and an activator in adult reproduction remains elusive. Here, we use the insect Locusta migratoria to dissect the molecular mechanism by which Kr-h1 functions as activator and repressor at these distinct developmental stages. Results: We report that the kinase PKCα triggers Kr-h1 phosphorylation at the amino acid residue Ser154, a step essential for its dual functions. During juvenile stage, phosphorylated Kr-h1 recruits a corepressor, C-terminal binding protein (CtBP). The complex of phosphorylated Kr-h1 and CtBP represses the transcription of Ecdysone induced protein 93F (E93) and consequently prevents the juvenile-to-adult transition. In adult insects, phosphorylated Kr-h1 recruits a coactivator, CREB-binding protein (CBP), and promotes vitellogenesis by inducing the expression of Ribosomal protein L36. Furthermore, Kr-h1 phosphorylation with the concomitant inhibition of E93 transcription is evolutionarily conserved across insect orders. Conclusion: Our results suggest that Kr-h1 phosphorylation is indispensable for the recruitment of transcriptional cofactors, and for its anti-metamorphic and vitellogenic actions in insects. Our data shed new light on the understanding of Kr-h1 regulation and function in JH-regulated insect metamorphosis and reproduction. [ABSTRACT FROM AUTHOR]

Gene transcription is strictly controlled by transcriptional complexes, which are assemblies of transcription factors, transcriptional regulators, and co-regulators. Mammalian genomes encode two C-terminal-binding proteins (CtBPs), CtBP1 and CtBP2, which are both well-known transcriptional corepressors of oncogenic processes. Their overexpression in tumors is associated with malignant behavior, such as uncontrolled cell proliferation, migration, and invasion, as well as with an increase in the epithelial-mesenchymal transition. CtBPs coordinate with other transcriptional regulators, such as histone deacetylases (HDACs) and histone acetyltransferases (p300 and CBP [CREBP-binding protein]) that contain the PXDLS motif, and with transcription factors to assemble transcriptional complexes that dock onto the promoters of genes to initiate gene transcription. Emerging evidence suggests that CtBPs function as both corepressors and coactivators in different biological processes ranging from apoptosis to inflammation and osteogenesis. Therapeutic targeting of CtBPs or the interactions required to form transcriptional complexes has also shown promising effects in preventing disease progression. This review summarizes the most recent progress in the study of CtBP functions and therapeutic inhibitors in different biological processes. This knowledge may enable a better understanding of the complexity of the roles of CtBPs, while providing new insights into therapeutic strategies that target CtBPs. [ABSTRACT FROM AUTHOR]

Background: Cisd1 and Cisd2 proteins share very similar structures with an N-terminal membrane-anchoring domain and a C-terminal cytosolic domain containing an iron-cluster binding domain and ending with a C-terminal KKxx sequence. Despite sharing a similar structure, Cisd1 and Cisd2 are anchored to different compartments: mitochondria for Cisd1 and endoplasmic reticulum for Cisd2. The aim of this study was to identify the protein motifs targeting Cisd2 to the ER and ensuring its retention in this compartment. Results: We used new recombinant antibodies to localize Cisd1 and Cisd2 proteins, as well as various protein chimeras. Cisd2 is targeted to the ER by its N-terminal sequence. It is then retained in the ER by the combined action of a C-terminal COPI-binding KKxx ER retrieval motif, and of an ER-targeting transmembrane domain. As previously reported for Cisd1, Cisd2 can alter the morphology of the compartment in which it accumulates. Conclusion: Although they share a very similar structure, Cisd1 and Cisd2 use largely different intracellular targeting motifs to reach their target compartment (mitochondria and endoplasmic reticulum, respectively). [ABSTRACT FROM AUTHOR]

Phytohormone auxin plays a fundamental role in plant growth and defense against pathogens. However, how auxin signalling is regulated during virus infection in plants remains largely unknown. Auxin/indole‐3‐acetic acid (Aux/IAA) is the repressor of auxin signalling and can be recognized by an F‐box protein transport inhibitor response 1 (TIR1). Ubiquitination and degradation of Aux/IAA by SKP1‐Cullin‐F‐boxTIR1 (SCFTIR1) complex can trigger auxin signalling. Here, with an emerging important plant virus worldwide, we showed that tomato chlorosis virus (ToCV) infection or stable transgenic overexpression of its p22 protein does not alter auxin accumulation level but significantly decreases the expression of auxin signalling–responsive genes, suggesting that p22 can attenuate host auxin signalling. Further, p22 could bind the C‐terminal of SKP1.1 and compete with TIR1 to interfere with the SCFTIR1 complex assembly, leading to a suppression of Aux/IAA degradation. Silencing and over‐expression assays suggested that both NbSKP1.1 and NbTIR1 suppress ToCV accumulation and disease symptoms. Altogether, ToCV p22 disrupts the auxin signalling through destabilizing SCFTIR1 by interacting with the C‐terminal of NbSKP1.1 to promote ToCV infection. Our findings uncovered a previously unknown molecular mechanism employed by a plant virus to manipulate SCF complex–mediated ubiquitin pathway and to reprogram auxin signalling for efficient infection. Tomato chlorosis virus–encoded p22 protein can bind to the C‐terminal domain of SKP1 to interfere with the SCFTIR1 complex assembly, leading to suppressed auxin signalling and enhanced viral infection. Our findings unveil a new mechanism used by a plant virus to subvert ubiquitination to enhance infection. [ABSTRACT FROM AUTHOR]

The homologous to E6AP C-terminus (HECT) domain and RCC1-like domain-containing (HERC) proteins can function as tumour suppressors and as oncogenes, depending on the cancer type. However, the expression patterns of HERCs in colorectal cancer (CRC) cells are unclear. Here, we show that only HERC1 and HERC5 are downregulated in CRC tumours, and we focus our study on revealing HERC5-mediating signalling because the change in downregulation is much more obvious for HERC5 than for HERC1. We demonstrate that HERC5 recruits an adaptor protein, CREB-binding protein, to ubiquitinate C-terminal binding protein 1 (CtBP1) in non-cancerous colon cells. The downregulation of HERC5 in CRC cells attenuates the ubiquitination of CtBP1, which then accumulates and assembles into a transcriptional complex with histone deacetylase 1 and a transcription factor c-MYC. This transcriptional complex binds to the promoters of three proapoptotic genes, Bcl2 associated X (BAX), Bcl2 interacting killer (BIK) and p53upregulated modulator of apoptosis (PUMA), and inhibits their expression, thereby suppressing apoptotic signalling and promoting tumourigenesis. Overexpression of HERC5 , downregulation of CtBP1 or blocking of the CtBP1 function with its inhibitors (NSC95397 and 4-methylthio-2-oxobutyric acid [MTOB]) significantly prevents CRC cell proliferation in vitro and tumour growth in vivo. Combining NSC95397 (or MTOB) with chemotherapeutic drugs (oxaliplatin or capecitabine) gives a much stronger inhibition of cell proliferation and tumour growth compared with their single treatments. Collectively, our results reveal that downregulation of HERC5 E3 ligase attenuates the ubiquitination of CtBP1 to inhibit apoptosis. Therefore, CtBP1 may be a promising target in CRC chemotherapy. [ABSTRACT FROM AUTHOR]

Enforcing differentiation of cancer stem cells is considered as a potential strategy to sensitize colorectal cancer cells to irradiation and chemotherapy. Activation of the unfolded protein response, due to endoplasmic reticulum (ER) stress, causes rapid stem cell differentiation in normal intestinal and colon cancer cells. We previously found that stem cell differentiation was mediated by a Protein kinase R-like ER kinase (PERK) dependent arrest of mRNA translation, resulting in rapid protein depletion of WNT-dependent transcription factor c-MYC. We hypothesize that ER stress dependent stem cell differentiation may rely on the depletion of additional transcriptional regulators with a short protein half-life that are rapidly depleted due to a PERK-dependent translational pause. Using a novel screening method, we identify novel transcription factors that regulate the intestinal stem cell fate upon ER stress. ER stress was induced in LS174T cells with thapsigargin or subtilase cytotoxin (SubAB) and immediate alterations in nuclear transcription factor activity were assessed by the CatTFRE assay in which transcription factors present in nuclear lysate are bound to plasmid DNA, co-extracted and quantified using mass-spectrometry. The role of altered activity of transcription factor CtBP2 was further examined by modification of its expression levels using CAG-rtTA3-CtBP2 overexpression in small intestinal organoids, shCtBP2 knockdown in LS174T cells, and familial adenomatous polyposis patient-derived organoids. CtBP2 overexpression organoids were challenged by ER stress and ionizing irradiation. We identified a unique set of transcription factors with altered activation upon ER stress. Gene ontology analysis showed that transcription factors with diminished binding were involved in cellular differentiation processes. ER stress decreased CtBP2 protein expression in mouse small intestine. ER stress induced loss of CtBP2 expression which was rescued by inhibition of PERK signaling. CtBP2 was overexpressed in mouse and human colorectal adenomas. Inducible CtBP2 overexpression in organoids conferred higher clonogenic potential, resilience to irradiation-induced damage and a partial rescue of ER stress-induced loss of stemness. Using an unbiased proteomics approach, we identified a unique set of transcription factors for which DNA-binding activity is lost directly upon ER stress. We continued investigating the function of co-regulator CtBP2, and show that CtBP2 mediates ER stress-induced loss of stemness which supports the intestinal stem cell state in homeostatic stem cells and colorectal cancer cells. [ABSTRACT FROM AUTHOR]

The recruitment and subsequent polarization of inflammatory monocytes/macrophages in the perivascular regions of pulmonary arteries is a key feature of pulmonary hypertension (PH). However, the mechanisms driving macrophage polarization within the adventitial microenvironment during PH progression remain unclear. We previously established that reciprocal interactions between fibroblasts and macrophages are essential in driving the activated phenotype of both cell types although the signals involved in these interactions remain undefined. We sought to test the hypothesis that adventitial fibroblasts produce a complex array of metabolites and proteins that coordinately direct metabolomic and transcriptomic re-programming of naïve macrophages to recapitulate the pathophysiologic phenotype observed in PH. Media conditioned by pulmonary artery adventitial fibroblasts isolated from pulmonary hypertensive (PH-CM) or age-matched control (CO-CM) calves were used to activate bone marrow derived macrophages. RNA-Seq and mass spectrometry-based metabolomics analyses were performed. Fibroblast conditioned medium from patients with idiopathic pulmonary arterial hypertension or controls were used to validate transcriptional findings. The microenvironment was targeted in vitro using a fibroblast-macrophage co-culture system and in vivo in a mouse model of hypoxia-induced PH. Both CO-CM and PH-CM actively, yet distinctly regulated macrophage transcriptomic and metabolomic profiles. Network integration revealed coordinated rewiring of pro-inflammatory and pro-remodeling gene regulation in concert with altered mitochondrial and intermediary metabolism in response to PH-CM. Pro-inflammation and metabolism are key regulators of macrophage phenotype in vitro , and are closely related to in vivo flow sorted lung interstitial/perivascular macrophages from hypoxic mice. Metabolic changes are accompanied by increased free NADH levels and increased expression of a metabolic sensor and transcriptional co-repressor, C-terminal binding protein 1 (CtBP1), a mechanism shared with adventitial PH-fibroblasts. Targeting the microenvironment created by both cell types with the CtBP1 inhibitor MTOB, inhibited macrophage pro-inflammatory and metabolic re-programming both in vitro and in vivo. In conclusion, coordinated transcriptional and metabolic reprogramming is a critical mechanism regulating macrophage polarization in response to the complex adventitial microenvironment in PH. Targeting the adventitial microenvironment can return activated macrophages toward quiescence and attenuate pathological remodeling that drives PH progression. [ABSTRACT FROM AUTHOR]

The reticulon family of integral membrane proteins are conserved across all eukaryotes and typically localize to the endoplasmic reticulum (ER), where they are involved in generating highly-curved tubules. We recently demonstrated that Reticulon-like protein B13 (RTNLB13) from Arabidopsis thaliana contains a curvature-responsive amphipathic helix (APH) important for the proteins' ability to induce curvature in the ER membrane, but incapable of generating curvature by itself. We suggested it acts as a feedback element, only folding/binding once a sufficient degree of curvature has been achieved, and stabilizes curvature without disrupting the bilayer. However, it remains unclear whether this is unique to RTNLB13 or is conserved across all reticulons—to date, experimental evidence has only been reported for two reticulons. Here we used biophysical methods to characterize a minimal library of putative APH peptides from across the 21 A. thaliana isoforms. We found that reticulons with the closest evolutionary relationship to RTNLB13 contain curvature-sensing APHs in the same location with sequence conservation. Our data reveal that a more distantly-related branch of reticulons developed a ~ 20-residue linker between the transmembrane domain and APH. This may facilitate functional flexibility as previous studies have linked these isoforms not only to ER remodeling but other cellular activities. [ABSTRACT FROM AUTHOR]

Germ cells in Drosophila melanogaster need intrinsic factors along with somatic signals to activate proper sexual programs. A key factor for male germline sex determination is PHD finger protein 7 (Phf7), a histone reader expressed in the male germline that can trigger sex reversal in female germ cells and is also important for efficient spermatogenesis. Here we find that the evolutionarily novel C-terminus in Phf7 is necessary to turn on the complete male program in the early germline of D. melanogaster, suggesting that this domain may have been uniquely acquired to regulate sexual differentiation. We further looked for genes regulated by Phf7 related to sex determination in the embryonic germline by transcriptome profiling of FACS-purified embryonic gonads. One of the genes positively-regulated by Phf7 in the embryonic germline was an HP1family member, Heterochromatin Protein 1D3 chromoshadow domain (HP1D3csd). We find that this gene is needed for Phf7 to induce male-like development in the female germline, indicating that HP1D3csd is an important factor acting downstream of Phf7 to regulate germline masculinization. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV) infection is associated with the development of specific types of lymphoma and some epithelial cancers. EBV infection of resting B-lymphocytes in vitro drives them to proliferate as lymphoblastoid cell lines (LCLs) and serves as a model for studying EBV lymphomagenesis. EBV nuclear antigen 3C (EBNA3C) is one of the genes required for LCL growth and previous work has suggested that suppression of the CDKN2A encoded tumor suppressor p16INK4A and possibly p14ARF is central to EBNA3C's role in this growth transformation. To directly assess whether loss of p16 and/or p14 was sufficient to explain EBNA3C growth effects, we used CRISPR/Cas9 to disrupt specific CDKN2A exons in EBV transformed LCLs. Disruption of p16 specific exon 1α and the p16/p14 shared exon 2 were each sufficient to restore growth in the absence of EBNA3C. Using EBNA3C conditional LCLs knocked out for either exon 1α or 2, we identified EBNA3C induced and repressed genes. By trans-complementing with EBNA3C mutants, we determined specific genes that require EBNA3C interaction with RBPJ or CtBP for their regulation. Unexpectedly, interaction with the CtBP repressor was required not only for repression, but also for EBNA3C induction of many host genes. Contrary to previously proposed models, we found that EBNA3C does not recruit CtBP to the promoters of these genes. Instead, our results suggest that CtBP is bound to these promoters in the absence of EBNA3C and that EBNA3C interaction with CtBP interferes with the repressive function of CtBP, leading to EBNA3C mediated upregulation. Author summary: Epstein-Barr virus (EBV) is a gammaherpesvirus that establishes lifelong infection in about 95% of adult humans. EBV infection is usually benign, but can rarely result in several different malignancies, particularly lymphomas. EBV infection of resting B-lymphocytes in the laboratory drives them to proliferate as lymphoblastoid cell lines (LCLs), a model for EBV lymphomagenesis. In this manuscript we study how one EBV protein expressed in LCLs, EBNA3C, contributes to B lymphocyte transformation. Prior work has established that EBNA3C turns off the CDKN2A gene, but there is disagreement regarding the relative importance of silencing the two CDKN2A gene products: p14 and p16. Using a CRISPR/Cas9 gene editing strategy we confirm that p16 knock-out rescues LCL growth in the absence of EBNA3C even in the presence of wildtype p14. We then use these knock-out LCLs to identify EBNA3C regulated genes and uncover extensive growth-independent changes in B lymphocytes due to the EBNA3C transcription factor. We also discover an unexpected role for the CtBP repressor protein in EBNA3C gene upregulation. Contrary to prior models, we do not observe CtBP recruitment to target genes by EBNA3C. Instead, our data are consistent with EBNA3C interfering with the ability of pre-bound CtBP to repress genes. [ABSTRACT FROM AUTHOR]

C-terminal binding proteins (CtBPs) are transcriptional modulators that can regulate gene expression through the recruitment of a corepressor complex composed of chromatin-modifying enzymes and transcriptional factors. In the brain, CtBPs have been described as regulators of cell proliferation, differentiation, and survival. Nevertheless, the role of CtBPs on postnatal neural stem cells (NSCs) fate is not known yet. Herein, we evaluate the expression and functions of CtBPs in postnatal NSCs from the subventricular zone (SVZ). We found that CtBPs were expressed in immature/progenitor cells, neurons and glial cells in the SVZ niche. Using the CtBPs modulator 4-methylthio 2-oxobutyric acid (MTOB), our results showed that 1 mM of MTOB induced cell death, while 5, 25, and 50 μM increased the number of proliferating neuroblasts, mature neurons, and oligodendrocytes. Interestingly, it also increased the dendritic complexity of immature neurons. Altogether, our results highlight CtBPs putative application for brain regenerative applications.

Light-induction of an anionic semiquinone (SQ) flavin radical in Drosophila cryptochrome (dCRY) alters the dCRY conformation to promote binding and degradation of the circadian clock protein Timeless (TIM). Specific peptide ligation with sortase A attaches a nitroxide spin-probe to the dCRY C-terminal tail (CTT) while avoiding deleterious side reactions. Pulse dipolar electron-spin resonance spectroscopy from the CTT nitroxide to the SQ shows that flavin photoreduction shifts the CTT ~1 nm and increases its motion, without causing full displacement from the protein. dCRY engineered to form the neutral SQ serves as a dark-state proxy to reveal that the CTT remains docked when the flavin ring is reduced but uncharged. Substitutions of flavin-proximal His378 promote CTT undocking in the dark or diminish undocking in the light, consistent with molecular dynamics simulations and TIM degradation activity. The His378 variants inform on recognition motifs for dCRY cellular turnover and strategies for developing optogenetic tools. Chandrasekaran et al. engineered the Drosophila circadian clock protein Cryptochrome (dCRY) to form the neutral semiquinone, which serves as a dark-state proxy. They find that the C-terminal tail of dCRY remains docked when the flavin ring is reduced but uncharged. dCRY His378 variants provide insights into the recognition motifs for dCRY turnover and strategies for optogenetic tools. [ABSTRACT FROM AUTHOR]

Marek's disease virus (MDV) encodes a basic-leucine zipper (BZIP) protein, Meq, which is considered the major MDV oncoprotein. It has been reported that the oncogenicity of Meq is associated with its interaction with C-terminal binding protein 1 (CtBP), which is also an interaction partner of Epstein-Barr virus encoded EBNA3A and EBNA3C oncoproteins. Since both EBNA3C and CtBP interact with histone deacetylase 1 (HDAC1) and HDAC2, we examined whether Meq shares this interaction with chicken HDAC1 (chHDAC1) and chHDAC2. Using confocal microscopy analysis, we show that Meq co-localizes with chHDAC1 and chHDAC2 in the nuclei of MDV lymphoblastoid tumor cells. In addition, immunoprecipitation assays demonstrate that Meq interacts with chHDAC1 and chHDAC2 in transfected cells and MDV lymphoblastoid tumor cells. Using deletion mutants, interaction domains were mapped to the N-terminal dimerization domain of chHDAC1 and chHDAC2, and the BZIP domain of Meq. Our results further demonstrate that this interaction mediates the degradation of chHDAC1 and chHDAC2 via the proteasome dependent pathway. In addition, our results show that Meq also induces the reduction of global ubiquitinated proteins through a proteasome dependent pathway. In conclusion, our results provide evidence that Meq interacts with chHDAC1 and chHDAC2, and induces their proteasome dependent degradation. [ABSTRACT FROM AUTHOR]

Summary: The apparent antagonism between salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signalling resulting in trade‐offs between defence against (hemi)biotrophic and necrotrophic pathogens has been widely described across multiple plant species. However, the underlying mechanism remains to be fully established.The molecular and cellular functions of ANGUSTIFOLIA (AN) were characterised, and its role in regulating the pathogenic response was studied in Arabidopsis.We demonstrated that AN, a plant homologue of mammalian C‐TERMINAL BINDING PROTEIN (CtBP), antagonistically regulates plant resistance to the hemibiotrophic pathogen Pseudomonas syringae and the necrotrophic pathogen Botrytis cinerea. Consistent with phenotypic observations, transcription of genes involved in SA and JA/ET pathways was antagonistically regulated by AN. By interacting with another nuclear protein TYROSYL‐DNA PHOSPHODIESTERASE1 (TDP1), AN imposes transcriptional repression on MYB46, encoding a transcriptional activator of PHENYLALANINE AMMONIA‐LYASE (PAL) genes which are required for SA biosynthesis, while releasing TDP1‐imposed transcriptional repression on WRKY33, a master regulator of the JA/ET signalling pathway.These findings demonstrate that transcriptional co‐regulation of MYB46 and WRKY33 by AN mediates the coordination of SA and JA/ET pathways to optimise defences against (hemi)biotrophic and necrotrophic pathogens. [ABSTRACT FROM AUTHOR]

Background: This study aimed at probing into the effect of long non-coding RNA (lncRNA) C-terminal binding protein 1 antisense RNA 2 (CTBP1-AS2) on gastric cancer (GC) cell proliferation and apoptosis, and its regulatory function on miR-139-3p and MMP11. Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to examine the expressions of CTBP1-AS2, miR-139-3p and MMP11 mRNA in GC cell lines and clinical specimens. Cell counting kit-8 (CCK-8) assay, flow cytometry and EdU assay were conducted to examine the effects of CTBP1-AS2 and miR-139-3p on GC cell proliferation and apoptosis. Western blot was applied for detecting the expressions of Bax, Bcl-2 and MMP11. A lung metastasis mouse model was used to evaluate metastasis of GC cells in vivo. Bioinformatics, dual-luciferase report assay, RIP and RNA pull-down assays were utilized to validate the targeted relationship between CTBP1-AS2 and miR-139-3p as well as the targeting relationship between miR-139-3p and MMP11. Results: CTBP1-AS2 was highly expressed in GC, and its high expression was strongly associated with increased TNM stage, increased tumor size and low degree of differentiation of the tumor tissues. Meanwhile, CTBP1-AS2 promoted GC cell proliferation, metastasis and suppressed apoptosis, while miR-139-3p could weaken these effects. In addition, CTBP1-AS2 was identified as a molecular sponge for miR-139-3p, and MMP11 was verified as a target gene of CTBP1-AS2. CTBP1-AS2 could increase the expression of MMP11 via repressing miR-139-3p. Conclusion: CTBP1-AS2 promotes GC cells and inhibits apoptosis by regulating the miR-139-3p/MMP11 molecular axis. [ABSTRACT FROM AUTHOR]

Brain development requires precise regulation of axon outgrowth, guidance and termination by multiple signaling and adhesion molecules. How the expression of these neurodevelopmental regulators is transcriptionally controlled is poorly understood. The Caenorhabditis elegans SMD motor neurons terminate axon outgrowth upon sexual maturity and partially retract their axons during early adulthood. Here we show that C-terminal binding protein 1 (CTBP-1), a transcriptional corepressor, is required for correct SMD axonal development. Loss of CTBP-1 causes multiple defects in SMD axon development: premature outgrowth, defective guidance, delayed termination and absence of retraction. CTBP-1 controls SMD axon guidance by repressing the expression of SAX-7, an L1 cell adhesion molecule (L1CAM). CTBP-1-regulated repression is crucial because deregulated SAX-7/L1CAM causes severely aberrant SMD axons.We found that axonal defects caused by deregulated SAX-7/L1CAM are dependent on a distinct L1CAM, called LAD-2, which itself plays a parallel role in SMD axon guidance. Our results reveal that harmonization of L1CAM expression controls the development and maturation of a single neuron. [ABSTRACT FROM AUTHOR]

Adaptive responses to hypoxia are mediated by the hypoxia‐inducible factor (HIF) family of transcription factors. These responses include the upregulation of glycolysis to maintain ATP production. This also generates acidic metabolites, which require HIF‐induced carbonic anhydrase IX (CAIX) for their neutralisation. C‐terminal binding proteins (CtBPs) are coregulators of gene transcription and couple glycolysis with gene transcription due to their regulation by the glycolytic coenzyme NADH. Here, we find that experimental manipulation of glycolysis and CtBP function in breast cancer cells through multiple complementary approaches supports a hypothesis whereby the expression of known HIF‐inducible genes, and CAIX in particular, adapts to available glucose in the microenvironment through a mechanism involving CtBPs. This novel pathway promotes the survival of stem cell‐like cancer (SCLC) cells in hypoxia. [ABSTRACT FROM AUTHOR]

The pluripotency-associated transcriptional network is regulated by a core circuitry of transcription factors. The PR domain-containing protein PRDM14 maintains pluripotency by activating and repressing transcription in a target gene-dependent manner. However, the mechanisms underlying dichotomic switching of PRDM14-mediated transcriptional control remain elusive. Here, we identified C-terminal binding protein 1 and 2 (CtBP1 and CtBP2; generically referred to as CtBP1/2) as components of the PRDM14-mediated repressive complex. CtBP1/2 binding to PRDM14 depends on CBFA2T2, a core component of the PRDM14 complex. The loss of Ctbp1/2 impaired the PRDM14-mediated transcriptional repression required for pluripotency maintenance and transition from primed to naïve pluripotency. Furthermore, CtBP1/2 interacted with the PRC2 complexes, and the loss of Ctbp1/2 impaired Polycomb repressive complex 2 (PRC2) and H3K27me3 enrichment at target genes after Prdm14 induction. These results provide evidence that the target gene-dependent transcriptional activity of PRDM14 is regulated by partner switching to ensure the transition from primed to naïve pluripotency. [ABSTRACT FROM AUTHOR]

Background Epithelial–mesenchymal transition (EMT) plays a pivotal role in the progression of prostate cancer (PCa). However, little is known about genetic variants in the EMT pathway as predictors of aggressiveness, biochemical recurrence (BCR) and disease reclassification in localized PCa. Patients and methods In this multistage study, we evaluated 5186 single nucleotide polymorphisms (SNPs) from 264 genes related to EMT pathway to identify SNPs associated with PCa aggressiveness and BCR in the MD Anderson PCa (MDA-PCa) patient cohort (N = 1762), followed by assessment of the identified SNPs with disease reclassification in the active surveillance (AS) cohort (N = 392). Results In the MDA-PCa cohort, 312 SNPs were associated with high D'Amico risk (P < 0.05), among which, 14 SNPs in 10 genes were linked to BCR risk. In the AS cohort, 2 of 14 identified SNPs (rs76779889 and rs7083961) in C-terminal Binding Proteins 2 gene were associated with reclassification risk. The associations of rs76779889 with different endpoints were: D'Amico high versus low, odds ratio [95% confidence interval (CI)] = 2.89 (1.32–6.34), P = 0.008; BCR, hazard ratio (HR) (95% CI) = 2.88 (1.42–5.85), P = 0.003; and reclassification, HR (95% CI) = 2.83 (1.40–5.74), P = 0.004. For rs7083961, the corresponding risk estimates were: D'Amico high versus low, odds ratio (95% CI) = 1.69 (1.12–2.57), P = 0.013; BCR, HR (95% CI) = 1.87 (1.15–3.02), P = 0.011 and reclassification, HR (95% CI) = 1.72 (1.09–2.72), P = 0.020. There were cumulative effects of these two SNPs on modulating these endpoints. Conclusion Genetic variants in EMT pathway may influence the risks of localized PCa's aggressiveness, BCR and disease reclassification, suggesting their potential role in the assessment and management of localized PCa. [ABSTRACT FROM AUTHOR]

Melanoma represents the most serious type of skin cancer. Although recent years have seen advances using targeted and immunotherapies, most patients remain at high risk for tumor recurrence. Here we show that IRAK-M, a negative regulator of MyD88 signaling, is deficient or low in melanoma and expression levels correlate with patient survival. Inducing IRAK-M expression using genetic approaches or epigenetic modifiers initiates apoptosis by prompting its interaction with TRAF6 via IRAK-M's C-terminal domain. This complex recruits and degrades calpastatin which stimulates calpain activity and triggers caspase-3-dependent but caspase-8,−9-independent apoptosis. Using a drug screen, we identified compounds that induced IRAK-M expression. Administration of IRAK-M-inducing drugs reduced tumor growth in mice but was ineffective against IRAK-M knock-down tumors. These results uncover a previously uncharacterized apoptosis pathway, emphasize IRAK-M as a potential therapeutic target and suggest that the anticancer activity of certain drugs could do so through their ability to induce IRAK-M expression. Geng et al find that low levels of the anti-inflammatory molecule IRAK-M in melanoma correlates with reduced patient survival and that induced expression of IRAK-M induces caspase-3 dependent apoptosis. The identification of cytotoxic compounds associated with IRAK-M induction suggests a route to melanoma drug development. [ABSTRACT FROM AUTHOR]

Background: Adenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear. Methods: The dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding. Results: PMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells. Conclusions: Our data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation. [ABSTRACT FROM AUTHOR]

Npl4 is likely to be the most upstream factor recognizing Lys48-linked polyubiquitylated substrates in the proteasomal degradation pathway in yeast. Along with Ufd1, Npl4 forms a heterodimer (UN), and functions as a cofactor for the Cdc48 ATPase. Here, we report the crystal structures of yeast Npl4 in complex with Lys48-linked diubiquitin and with the Npl4-binding motif of Ufd1. The distal and proximal ubiquitin moieties of Lys48-linked diubiquitin primarily interact with the C-terminal helix and N-terminal loop of the Npl4 C-terminal domain (CTD), respectively. Mutational analysis suggests that the CTD contributes to linkage selectivity and initial binding of ubiquitin chains. Ufd1 occupies a hydrophobic groove of the Mpr1/Pad1 N-terminal (MPN) domain of Npl4, which corresponds to the catalytic groove of the MPN domain of JAB1/MPN/Mov34 metalloenzyme (JAMM)-family deubiquitylating enzyme. This study provides important structural insights into the polyubiquitin chain recognition by the Cdc48–UN complex and its assembly. The Lys48-linked polyubiquitin-mediated proteasomal degradation in yeast depends on Cdc48 and its cofactors Ufd1 and Npl4. Here, the authors present crystal structures of Npl4 bound to Lys48-linked diubiquitin and the Npl4-binding motif of Ufd1, providing insights into the reaction mechanism of the Cdc48- Ufd1/Npl4 complex. [ABSTRACT FROM AUTHOR]

C‐terminal binding protein 1 (CtBP1), a well‐known transcriptional corepressor, functions as an oncogene in multiple cancer types, including osteosarcoma, by modulating the transcription of many tumor suppressors, such as cadherin 1 (CDH1), phosphatase and tensin homolog (PTEN), Bcl2‐associated X (Bax), Bcl‐2‐interacting mediator (Bim), and cyclin‐dependent kinase inhibitor 1A (CDKN1A). However, it is still unclear how CtBP1 regulates the expression of these downstream targets. Here, we identified that CtBP1 is overexpressed in osteosarcoma cells and found that CtBP1 directly interacts with the transcription factor forkhead box O3 (FOXO3a) and the histone acetyltransferase p300 in vivo and in vitro. Through microarray analysis, we found that CtBP1 negatively regulates FOXO3a levels. In contrast to the CtBP1 level, the FOXO3a expression level was found to be significantly reduced in osteosarcoma cells. Knockdown of CtBP1 or overexpression of FOXO3a in U2OS cells resulted in different gene expression patterns, and the former caused upregulation of CtBP1 downstream target genes such as CDH1, PTEN, Bax, Bim, and CDKN1A, whereas the latter caused upregulation of Bax and Bim, but not CDH1, PTEN, and CDKN1A. Further analysis indicated that the CtBP1–p300–FOXO3a transcriptional complex specifically binds to the promoters of Bax and Bim. Inhibition of CtBP1 by the constitutive expression of Pep1‐E1AWT peptide in U2OS and OSA cells reversed oncogenic phenotypes, including colony formation, cellular proliferation, and migration, and limited tumor growth in vivo. Together our results demonstrated that the CtBP1–p300–FOXO3a transcriptional complex represses the expression of the apoptotic regulators Bax and Bim in human osteosarcoma cells and that targeting CtBP1‐mediated transcriptional events might be a potential therapeutic strategy for the osteosarcoma treatment. [ABSTRACT FROM AUTHOR]

Guanylate-binding proteins (GBPs) constitute a family of interferon-inducible guanosine triphosphatases (GTPases) that are key players in host defense against intracellular pathogens ranging from protozoa to bacteria and viruses. So far, human GBP1 and GBP5 as well as murine GBP2 (mGBP2) have been biochemically characterized in detail. Here, with murine GBP7 (mGBP7), a GBP family member with an unconventional and elongated C-terminus is analyzed. The present study demonstrates that mGBP7 exhibits a concentration-dependent GTPase activity and an apparent GTP turnover number of 20 min-1. In addition, fluorescence spectroscopy analyses reveal that mGBP7 binds GTP with high affinity (KD = 0.22 mM) and GTPase activity assays indicate that mGBP7 hydrolyzes GTP to GDP and GMP. The mGBP7 GTPase activity is inhibited by incubation with γ-phosphate analogs and a K51A mutation interfering with GTP binding. SEC-MALS analyses give evidence that mGBP7 forms transient dimers and that this oligomerization pattern is not influenced by the presence of nucleotides. Moreover, a structural model for mGBP7 is provided by homology modeling, which shows that the GTPase possesses an elongated C-terminal (CT) tail compared with the CaaX motif-containing mGBP2 and human GBP1. Molecular dynamics simulations indicate that this tail has transmembrane characteristics and, interestingly, confocal microscopy analyses reveal that the CT tail is required for recruitment of mGBP7 to the parasitophorous vacuole of Toxoplasma gondii. [ABSTRACT FROM AUTHOR]