RESUMEN
Numerous staple crops exhibit polyploidy and are difficult to genetically modify. However, recent advances in genome sequencing and editing have enabled polyploid genome engineering. The hexaploid black nightshade species Solanum nigrum has immense potential as a beneficial food supplement. We assembled its genome at the scaffold level. After functional annotations, we identified homoeologous gene sets, with similar sequence and expression profiles, based on comparative analyses of orthologous genes with close diploid relatives Solanum americanum and S. lycopersicum. Using CRISPR-Cas9-mediated mutagenesis, we generated various mutation combinations in homoeologous genes. Multiple mutants showed quantitative phenotypic changes based on the genotype, resulting in a broad-spectrum effect on the quantitative traits of hexaploid S. nigrum. Furthermore, we successfully improved the fruit productivity of Boranong, an orphan cultivar of S. nigrum suggesting that engineering homoeologous genes could be useful for agricultural improvement of polyploid crops.
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Productos Agrícolas , Poliploidía , Secuencia de Bases , Mapeo Cromosómico/métodos , Mutación , Fenotipo , Productos Agrícolas/genética , Genoma de Planta/genética , Edición GénicaRESUMEN
CP2c, also known as TFCP2, α-CP2, LSF, and LBP-1c, is a prototypic member of the transcription factor (TF) CP2 subfamily involved in diverse ubiquitous and tissue/stage-specific cellular processes and in human malignancies including cancer. Despite its importance, many fundamental regulatory mechanisms of CP2c are still unclear. Here, we uncover unprecedented structural and functional aspects of CP2c using DSP crosslinking and Western blot in addition to conventional methods. We found that a monomeric form of a CP2c homotetramer (tCP2c; [C4]) binds to the known CP2c-binding DNA motif (CNRG-N(5~6)-CNRG), whereas a dimeric form of a CP2c, CP2b, and PIAS1 heterohexamer ([C2B2P2]2) binds to the three consecutive CP2c half-sites or two staggered CP2c binding motifs, where the [C4] exerts a pioneering function for recruiting the [C2B2P2]2 to the target. All CP2c exists as a [C4], or as a [C2B2P2]2 or [C2B2P2]4 in the nucleus. Importantly, one additional cytosolic heterotetrameric CP2c and CP2a complex, ([C2A2]), exerts some homeostatic regulation of the nuclear complexes. These data indicate that these findings are essential for the transcriptional regulation of CP2c in cells within relevant timescales, providing clues not only for the transcriptional regulation mechanism by CP2c but also for future therapeutics targeting CP2c function.
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Proteínas de Unión al ADN , Factores de Transcripción , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Humanos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.
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Proteínas de Unión al ADN/metabolismo , Eritropoyesis/fisiología , Globinas/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Diferenciación Celular , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Células Eritroides/citología , Eritropoyesis/genética , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Hemoglobinas/biosíntesis , Hemoglobinas/genética , Humanos , Masculino , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones Endogámicos BALB C , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genéticaRESUMEN
Intrinsically disordered proteins exist as highly dynamic conformational ensembles of diverse forms. However, the majority of virtual screening only focuses on proteins with defined structures. This means that computer-aided drug discovery is restricted. As a breakthrough, understanding the structural characteristics of intrinsically disordered proteins and its application can open the gate for unrestricted drug discovery. First, we segmented the target disorder-to-order transition region into a series of overlapping 20-amino-acid-long peptides. Folding prediction generated diverse conformations of these peptides. Next, we applied molecular docking, new evaluation score function, and statistical analysis. This approach successfully distinguished known compounds and their corresponding binding regions. Especially, Myc proto-oncogene protein (MYC) inhibitor 10058F4 was well distinguished from others of the chemical compound library. We also studied differences between the two Methyl-CpG-binding domain protein 2 (MBD2) inhibitors (ABA (2-amino-N-[[(3S)-2,3-dihydro-1,4-benzodioxin-3-yl]methyl]-acetamide) and APC ((R)-(3-(2-Amino-acetylamino)-pyrrolidine-1-carboxylic acid tert-butyl ester))). Both compounds bind MBD2 through electrostatic interaction behind its p66α-binding site. ABA is also able to bind p66α through electrostatic interaction behind its MBD2-binding site while APC-p66α binding was nonspecific. Therefore, structural heterogeneity mimicking of the disorder-to-order transition region at the peptide level and utilization of the new docking score function represent a useful approach that can efficiently discriminate compounds for expanded virtual screening toward intrinsically disordered proteins.
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Proteínas de Unión al ADN/química , Descubrimiento de Drogas , Proteínas Intrínsecamente Desordenadas/química , Simulación del Acoplamiento Molecular , Bibliotecas de Moléculas Pequeñas/química , Humanos , Unión Proteica , Proto-Oncogenes Mas , Electricidad EstáticaRESUMEN
Yin Yang 1 (YY1) is a transcription factor that regulates epigenetic pathways and protein modifications. CP2c is a transcription factor that functions as an oncogene to regulate cell proliferation. YY1 is known to interact with CP2c to suppress CP2c's transcriptional activity. This study aimed to investigate YY1 and CP2c expression in breast cancer and prognostic implications. In this study, YY1 and CP2c expression was evaluated using immunohistochemical staining, Western blot and RT-PCR assays. Of 491 patients with primary breast cancer, 138 patients showed YY1 overexpression. Luminal subtype and early stage were associated with overexpression (p < 0.001). After a median follow-up of 68 months, YY1 overexpression was found to be associated with a better prognosis (disease-free survival rates of 92.0% vs. 79.2%, p = 0.014). In Cox proportional hazards model, YY1 overexpression functioned as an independent prognostic factor after adjustment of hormone receptor/HER2 status and tumor size (hazard ratio of 0.50, 95% CI 0.26-0.98, p = 0.042). Quantitative analysis of YY1 and CP2c protein expression in tumors revealed a negative correlation between them. In conclusion, YY1 overexpression is a favorable prognostic biomarker in patients with breast cancer, and it has a negative correlation with CP2c at the protein level.
RESUMEN
Proteins play a crucial role in many biological processes, where their interaction with other proteins are integral. Abnormal protein-protein interactions (PPIs) have been linked to various diseases including cancer, and thus targeting PPIs holds promise for drug development. However, experimental confirmation of the peculiarities of PPIs is challenging due to their dynamic and transient nature. As a complement to experimental technologies, multiple computational molecular docking (MD) methods have been developed to predict the structures of protein-protein complexes and their dynamics, still requiring further improvements in several issues. Here, we report an improved MD method, namely three-software docking (3SD), by employing three popular protein-peptide docking software (CABS-dock, HPEPDOCK, and HADDOCK) in combination to ensure constant quality for most targets. We validated our 3SD performance in known protein-peptide interactions (PpIs). We also enhanced MD performance in proteins having intrinsically disordered regions (IDRs) by applying the modified 3SD strategy, the three-software docking after removing random coiled IDR (3SD-RR), to the comparable crystal PpI structures. At the end, we applied 3SD-RR to the AlphaFold2-predicted receptors, yielding an efficient prediction of PpI pose with high relevance to the experimental data regardless of the presence of IDRs or the availability of receptor structures. Our study provides an improved solution to the challenges in studying PPIs through computational docking and has the potential to contribute to PPIs-targeted drug discovery. SIGNIFICANCE STATEMENT: Protein-protein interactions (PPIs) are integral to life, and abnormal PPIs are associated with diseases such as cancer. Studying protein-peptide interactions (PpIs) is challenging due to their dynamic and transient nature. Here we developed improved docking methods (3SD and 3SD-RR) to predict the PpI poses, ensuring constant quality in most targets and also addressing issues like intrinsically disordered regions (IDRs) and artificial intelligence-predicted structures. Our study provides an improved solution to the challenges in studying PpIs through computational docking and has the potential to contribute to PPIs-targeted drug discovery.
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Inteligencia Artificial , Neoplasias , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Proteínas/química , Programas Informáticos , Péptidos/químicaRESUMEN
Transcription factor CP2c (also known as TFCP2, α-CP2, LSF, and LBP-1c) is involved in diverse ubiquitous and tissue/stage-specific cellular processes and in human malignancies such as cancer. Despite its importance, many fundamental regulatory mechanisms of CP2c are still unclear. Here, we uncover an unprecedented mechanism of CP2c degradation via a previously unidentified SUMO1/PSME3/20S proteasome pathway and its biological meaning. CP2c is SUMOylated in a SUMO1-dependent way, and SUMOylated CP2c is degraded through the ubiquitin-independent PSME3 (also known as REGγ or PA28)/20S proteasome system. SUMOylated PSME3 could also interact with CP2c to degrade CP2c via the 20S proteasomal pathway. Moreover, precisely timed degradation of CP2c via the SUMO1/PSME3/20S proteasome axis is required for accurate progression of the cell cycle. Therefore, we reveal a unique SUMO1-mediated uncanonical 20S proteasome degradation mechanism via the SUMO1/PSME3 axis involving mutual SUMO-SIM interaction of CP2c and PSME3, providing previously unidentified mechanistic insights into the roles of dynamic degradation of CP2c in cell cycle progression.
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Neoplasias , Complejo de la Endopetidasa Proteasomal , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Sumoilación , Citoplasma/metabolismo , Neoplasias/metabolismo , Ciclo Celular , Proteínas de Unión al ADN/metabolismoRESUMEN
Despite advances in precision oncology, cancer remains a global public health issue. In this report, proof-of-principle evidence is presented that a cell-penetrable peptide (ACP52C) dissociates transcription factor CP2c complexes and induces apoptosis in most CP2c oncogene-addicted cancer cells through transcription activity-independent mechanisms. CP2cs dissociated from complexes directly interact with and degrade YY1, leading to apoptosis via the MDM2-p53 pathway. The liberated CP2cs also inhibit TDP2, causing intrinsic genome-wide DNA strand breaks and subsequent catastrophic DNA damage responses. These two mechanisms are independent of cancer driver mutations but are hindered by high MDM2 p60 expression. However, resistance to ACP52C mediated by MDM2 p60 can be sensitized by CASP2 inhibition. Additionally, derivatives of ACP52C conjugated with fatty acid alone or with a CASP2 inhibiting peptide show improved pharmacokinetics and reduced cancer burden, even in ACP52C-resistant cancers. This study enhances the understanding of ACP52C-induced cancer-specific apoptosis induction and supports the use of ACP52C in anticancer drug development.
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Proteínas de Unión al ADN , Neoplasias , Humanos , Proteínas de Unión al ADN/genética , Neoplasias/genética , Mutaciones Letales Sintéticas , Medicina de Precisión , Factores de Transcripción/genética , Péptidos , Hidrolasas Diéster Fosfóricas/genéticaRESUMEN
Melanin is considered a bio-inspired dermo-cosmetic component due to its high UV absorption and antioxidant activity. Among various melanin sources, fungal melanin is a promising candidate for sunscreen because of its sustainability and scalability; however, quantitative assessment of its function has not yet been sufficiently explored. In this study, melanin samples derived from Amorphotheca resinae were prepared, followed by the evaluation of their sunscreen performance, antioxidant activity, and cytotoxicity. Melanin-blended cream was prepared by blending a melanin suspension and a pure cream. The cream showed an in vitro sun protection factor value of 2.5 when the pigment content was 5%. The cream showed a critical wavelength of approximately 388 nm and a UVA/UVB ratio of more than 0.81, satisfying the broad-spectrum sunscreen requirement. Oxygen radical absorbance capacity assays indicated that fungal melanin had antioxidant activity similar to ascorbic acid but higher than reduced glutathione. Fungal melanin had no statistically significant cytotoxicity to human keratinocyte cell lines until 72 h of exposure, even at a concentration of 4 mg mL-1. Consequently, melanin pigment can be used as a biocompatible broad-spectrum sunscreen with high antioxidant activity and as a practical alternative in dermo-cosmetic formulations.
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Stemness and epithelial-mesenchymal transition (EMT) are two fundamental characteristics of metastasis that are controlled by diverse regulatory factors, including transcription factors. Compared with other subtypes of breast cancer, basal-type or triple-negative breast cancer (TNBC) has high frequencies of tumor relapse. However, the role of alpha-globin transcription factor CP2 (TFCP2) has not been reported as an oncogenic driver in those breast cancers. Here, we show that TFCP2 is a potent factor essential for EMT, stemness, and metastasis in breast cancer. TFCP2 directly bound promoters of EGF and TGFα to regulate their expression and stimulate autocrine signaling via EGFR. These findings indicate that TFCP2 is a new antimetastatic target and reveal a novel regulatory mechanism in which a positive feedback loop comprising EGF/TGFα and AKT can control malignant breast cancer progression. SIGNIFICANCE: TFCP2 is a new antimetastatic target that controls TNBC progression via a positive feedback loop between EGF/TGFα and the AKT signaling axis.
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Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Receptores ErbB/metabolismo , Retroalimentación Fisiológica , Femenino , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Células MCF-7 , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Células Madre Neoplásicas , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
Chromatin remodeling, including histone modification, chromatin (un)folding, and nucleosome remodeling, is a significant transcriptional regulation mechanism. By these epigenetic modifications, transcription factors and their regulators are recruited to the promoters of target genes, and thus gene expression is controlled through either transcriptional activation or repression. The Mat1-mediated transcriptional repressor (MMTR)/DNA methyltransferase 1 (DNMT1)-associated protein (Dmap1) is a transcription corepressor involved in chromatin remodeling, cell cycle regulation, DNA double-strand break repair, and tumor suppression. The Tip60-p400 complex proteins, including MMTR/Dmap1, interact with the oncogene Myc in embryonic stem cells (ESCs). These proteins interplay with the stem cell-related proteome networks and regulate gene expressions. However, the detailed mechanisms of their functions are unknown. Here, we show that MMTR/Dmap1, along with other Tip60-p400 complex proteins, bind the promoters of differentiation commitment genes in mouse ESCs. Hence, MMTR/Dmap1 controls gene expression alterations during differentiation. Furthermore, we propose a novel mechanism of MMTR/Dmap1 function in early stage lineage commitment of mouse ESCs by crosstalk with the polycomb group (PcG) proteins. The complex controls histone mark bivalency and transcriptional poising of commitment genes. Taken together, our comprehensive findings will help better understand the MMTR/Dmap1-mediated transcriptional regulation in ESCs and other cell types.
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Linaje de la Célula , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas Represoras/metabolismo , Animales , Diferenciación Celular , Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Lisina/metabolismo , Lisina Acetiltransferasa 5/metabolismo , Metilación , Ratones , Ratones SCID , Modelos Biológicos , Complejo Represivo Polycomb 2/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Represoras/química , Transactivadores/metabolismoRESUMEN
Although intrinsically disordered protein regions (IDPRs) are commonly engaged in promiscuous protein-protein interactions (PPIs), using them as drug targets is challenging due to their extreme structural flexibility. We report a rational discovery of inhibitors targeting an IDPR of MBD2 that undergoes disorder-to-order transition upon PPI and is critical for the regulation of the Mi-2/NuRD chromatin remodeling complex (CRC). Computational biology was essential for identifying target site, searching for promising leads, and assessing their binding feasibility and off-target probability. Molecular action of selected leads inhibiting the targeted PPI of MBD2 was validated in vitro and in cell, followed by confirming their inhibitory effects on the epithelial-mesenchymal transition of various cancer cells. Identified lead compounds appeared to potently inhibit cancer metastasis in a murine xenograft tumor model. These results constitute a pioneering example of rationally discovered IDPR-targeting agents and suggest Mi-2/NuRD CRC and/or MBD2 as a promising target for treating cancer metastasis.
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Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas Intrínsecamente Desordenadas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Dominios Proteicos/efectos de los fármacos , Animales , Biología Computacional , Descubrimiento de Drogas/métodos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/antagonistas & inhibidores , Ratones , Modelos Moleculares , Metástasis de la Neoplasia/tratamiento farmacológico , Metástasis de la Neoplasia/prevención & control , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Murine erythroleukemia (MEL) cells are often employed as a model to dissect mechanisms of erythropoiesis and erythroleukemia in vitro. Here, an allograft model using MEL cells resulting in splenomegaly was established to develop a diagnostic model for isolation/quantification of metastatic cells, anti-cancer drug screening, and evaluation of the tumorigenic or metastatic potentials of molecules in vivo. In this animal model, circulating MEL cells from the blood stream were successfully isolated and quantified with an additional in vitro cultivation step. In terms of the molecular-pathological analysis, we were able to successfully evaluate the functional discrimination between methyl-CpG-binding domain 2 (Mbd2) and p66α in erythroid differentiation, and tumorigenic potential in spleen and blood stream of allograft model mice. In addition, we found that the number of circulating MEL cells in anti-cancer drug-treated mice was dose-dependently decreased. Our data demonstrate that the newly established allograft model is useful to dissect erythroleukemia pathologies and non-invasively provides valuable means for isolation of metastatic cells, screening of anti-cancer drugs, and evaluation of the tumorigenic potentials.
RESUMEN
Recent studies have demonstrated an oncogenic role of the transcription factor (TF) CP2c in hepatocellular carcinoma (HCC) based on a strong correlation between CP2c expression, tumor grade, and aggressiveness. We recently found that CP2c directly interacts with another TF, YY1, which is also overexpressed in multiple cancers, including HCC. To evaluate if these proteins are co-regulated in carcinogenesis, we analyzed the expression of CP2c and YY1 in HCC (n = 136) tissues and examined the correlation between their expression and clinicopathological characteristics of HCC. Receiver operating characteristic analysis exhibited the validity of CP2c and nuclear YY1 expression as a diagnostic factor in HCC tissues. High expression of CP2c was significantly correlated with patient age, and higher histological grade, American Joint Committee on Cancer (AJCC) stage, and small and large vessel invasion in HCC tissues, whereas high expression of nuclear YY1 was significantly associated with higher AJCC stage and small vessel invasion. In univariate and multivariate analyses, high expression of CP2c was significantly correlated with disease free survival (DFS), indicating that CP2c expression is an independent prognostic factor for DFS in HCC patients. Patients with high expression of both CP2c and nuclear YY1 usually had a shorter median survival time and worse DFS prognosis than other patients, suggesting that combined detection of CP2c and nuclear YY1 is a useful prognostic marker in HCC patients.