RESUMEN
Knowledge of how Mediator and TFIID cross-talk contributes to promoter-enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genome-wide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II preinitiation complex (PIC) at promoters, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by promoter capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect on Mediator integrity but led to a consistent twofold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but had no effect on the other PIC components. PCHi-C revealed no consistent effect of MED4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.
Asunto(s)
ARN Polimerasa II , Factor de Transcripción TFIID , Animales , Complejo Mediador/genética , Complejo Mediador/metabolismo , Ratones , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Factor de Transcripción TFIID/genética , Transcripción GenéticaRESUMEN
Chromobox homolog 3 (Cbx3/heterochromatin protein 1γ [HP1γ]) stimulates cell differentiation, but its mechanism is unknown. We found that Cbx3 binds to gene promoters upon differentiation of murine embryonic stem cells (ESCs) to neural progenitor cells (NPCs) and recruits the Mediator subunit Med26. RNAi knockdown of either Cbx3 or Med26 inhibits neural differentiation while up-regulating genes involved in mesodermal lineage decisions. Thus, Cbx3 and Med26 together ensure the fidelity of lineage specification by enhancing the expression of neural genes and down-regulating genes specific to alternative fates.
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Diferenciación Celular , Linaje de la Célula , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/citología , Regulación de la Expresión Génica , Complejo Mediador/metabolismo , Células-Madre Neurales/citología , Animales , Células Cultivadas , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Proteínas Cromosómicas no Histona/genética , Quinasa 8 Dependiente de Ciclina/genética , Quinasa 8 Dependiente de Ciclina/metabolismo , Células Madre Embrionarias/metabolismo , Complejo Mediador/genética , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Regiones Promotoras Genéticas , ARN Interferente Pequeño/genéticaRESUMEN
Dihydropyrimidinase (DHPase) plays a crucial role in pyrimidine degradation, showcasing a broad substrate specificity that extends beyond pyrimidine catabolism, hinting at additional roles for this ancient enzyme. In this study, we solved the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with the neurotransmitter γ-aminobutyric acid (GABA) at a resolution of 1.97 Å (PDB ID 8WQ9). Our structural analysis revealed two GABA binding sites in each monomer of PaDHPase. Interactions between PaDHPase and GABA molecules, involving residues within a contact distance of <4 Å, were examined. In silico analyses via PISA and PLIP software revealed hydrogen bonds formed between the side chain of Cys318 and GABA 1, as well as the main chains of Ser333, Ile335, and Asn337 with GABA 2. Comparative structural analysis between GABA-bound and unbound states unveiled significant conformational changes at the active site, particularly within dynamic loop I, supporting the conclusion that PaDHPase binds GABA through the loop-out mechanism. Building upon this molecular evidence, we discuss and propose a working model. The study expands the GABA interactome by identifying DHPase as a novel GABA-interacting protein and provides structural insight into the interaction between a dimetal center in the protein's active site and GABA. Further investigations are warranted to explore potential interactions of GABA with other DHPase-like proteins and to understand whether DHPase may have additional regulatory and physiological roles in the cell, extending beyond pyrimidine catabolism.
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Amidohidrolasas , Ácido gamma-Aminobutírico , Amidohidrolasas/química , Ácido gamma-Aminobutírico/metabolismo , Proteínas , Neurotransmisores , PirimidinasRESUMEN
Acetyl-CoAacyltransferase2 (ACAA2) is a key enzyme in the fatty acid oxidation pathway that catalyzes the final step of mitochondrial ß oxidation, which plays an important role in fatty acid metabolism. The expression of ACAA2 is closely related to the occurrence and malignant progression of tumors. However, the function of ACAA2 in ovarian cancer is unclear. The expression level and prognostic value of ACAA2 were analyzed by databases. Gain and loss of function were carried out to explore the function of ACAA2 in ovarian cancer. RNA-seq and bioinformatics methods were applied to illustrate the regulatory mechanism of ACAA2. ACAA2 overexpression promoted the growth, proliferation, migration, and invasion of ovarian cancer, and ACAA2 knockdown inhibited the malignant progression of ovarian cancer as well as the ability of subcutaneous tumor formation in nude mice. At the same time, we found that OGT can induce glycosylation modification of ACAA2 and regulate the karyoplasmic distribution of ACAA2. OGT plays a vital role in ovarian cancer as a function of oncogenes. In addition, through RNA-seq sequencing, we found that ACAA2 regulates the expression of DIXDC1. ACAA2 regulated the malignant progression of ovarian cancer through the WNT/ß-Catenin signaling pathway probably. ACAA2 is an oncogene in ovarian cancer and has the potential to be a target for ovarian cancer therapy.
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Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Ratones Desnudos , Neoplasias Ováricas , Femenino , Humanos , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Animales , Ratones , Línea Celular Tumoral , Movimiento Celular , Vía de Señalización Wnt , Pronóstico , Carcinogénesis/genéticaRESUMEN
Gene activation in metazoans is accompanied by the presence of histone variants H2AZ and H3.3 within promoters and enhancers. It is not known, however, what protein deposits H3.3 into chromatin or whether variant chromatin plays a direct role in gene activation. Here we show that chromatin containing acetylated H2AZ and H3.3 stimulates transcription in vitro. Analysis of the Pol II pre-initiation complex on immobilized chromatin templates revealed that the E1A binding protein p400 (EP400) was bound preferentially to and required for transcription stimulation by acetylated double-variant chromatin. EP400 also stimulated H2AZ/H3.3 deposition into promoters and enhancers and influenced transcription in vivo at a step downstream of the Mediator complex. EP400 efficiently exchanged recombinant histones H2A and H3.1 with H2AZ and H3.3, respectively, in a chromatin- and ATP-stimulated manner in vitro. Our data reveal that EP400 deposits H3.3 into chromatin alongside H2AZ and contributes to gene regulation after PIC assembly.
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Ensamble y Desensamble de Cromatina , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Acetilación , Adenosina Trifosfato/metabolismo , Sitios de Unión , Línea Celular Tumoral , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Genes Reporteros , Histonas/genética , Humanos , Interferencia de ARN , ARN Polimerasa II/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
We have successfully generated oligonucleotide aptamers (Apts) and monoclonal antibodies (mAbs) targeting the recombinant nucleocapsid (N) protein of SARS-CoV-2. Apts were obtained through seven rounds of systematic evolution of ligands by exponential enrichment (SELEX), while mAbs were derived from the 6F6E11 hybridoma cell line. Leveraging these Apts and mAbs, we have successfully devised two innovative and remarkably sensitive detection techniques for the rapid identification of SARS-CoV-2 N protein in nasopharyngeal samples: the enzyme-linked aptamer-antibody sandwich assay (ELAAA) and the hybrid lateral flow strip (hybrid-LFS). ELAAA exhibited an impressive detection limit of 0.1 ng/mL, while hybrid-LFS offered a detection range of 0.1 - 0.5 ng/mL. In the evaluation using ten nasopharyngeal samples spiked with known N protein concentrations, ELAAA demonstrated an average recovery rate of 92%. Additionally, during the assessment of five nasopharyngeal samples from infected individuals and ten samples from healthy volunteers, hybrid-LFS displayed excellent sensitivity and specificity. Our study introduces a novel and efficient on-site approach for SARS-CoV-2 detection in nasopharyngeal samples. The reliable hybrid Apt-mAb strategy not only advances virus diagnostic methods but also holds promise in combating the spread of related diseases.
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Aptámeros de Nucleótidos , COVID-19 , Humanos , SARS-CoV-2 , COVID-19/diagnóstico , Anticuerpos Monoclonales , Sensibilidad y EspecificidadRESUMEN
5-Fluorouracil (5-FU) stands as one of the most widely prescribed chemotherapeutics. Despite over 60 years of study, a systematic synopsis of how 5-FU binds to proteins has been lacking. Investigating the specific binding patterns of 5-FU to proteins is essential for identifying additional interacting proteins and comprehending their medical implications. In this review, an analysis of the 5-FU binding environment was conducted based on available complex structures. From the earliest complex structure in 2001 to the present, two groups of residues emerged upon 5-FU binding, classified as P- and R-type residues. These high-frequency interactive residues with 5-FU include positively charged residues Arg and Lys (P type) and ring residues Phe, Tyr, Trp, and His (R type). Due to their high occurrence, 5-FU binding modes were simplistically classified into three types, based on interactive residues (within <4 Å) with 5-FU: Type 1 (P-R type), Type 2 (P type), and Type 3 (R type). In summary, among 14 selected complex structures, 8 conform to Type 1, 2 conform to Type 2, and 4 conform to Type 3. Residues with high interaction frequencies involving the N1, N3, O4, and F5 atoms of 5-FU were also examined. Collectively, these interaction analyses offer a structural perspective on the specific binding patterns of 5-FU within protein pockets and contribute to the construction of a structural interactome delineating the associations of the anticancer drug 5-FU.
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Antineoplásicos , Fluorouracilo , Fluorouracilo/metabolismo , ProteínasRESUMEN
Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9's ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications.
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Tratamiento Farmacológico de COVID-19 , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Extractos Vegetales , Hojas de la Planta , SARS-CoV-2 , Humanos , Células A549 , Antivirales/farmacología , Antivirales/química , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , COVID-19/virología , COVID-19/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Simulación del Acoplamiento Molecular , Extractos Vegetales/farmacología , Extractos Vegetales/química , Hojas de la Planta/química , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidoresRESUMEN
OBJECTIVES: Myocarditis, a health-threatening heart disease, is attracting increasing attention. This systematic study was conducted to study the prevalence of disease through the trends of incidence, mortality, disability-adjusted life years (DALYs) over the last 30 years, which would be helpful for the policymakers to better the choices for reasonable decisions. METHODS: The global, regional, and national burdens of myocarditis from 1990-2019 were analyzed by using the 2019 Global Burden of Disease (GBD) database. This study on myocarditis produced new findings according to age, sex, and Social-Demographic Index (SDI) by investigating DALYs, age-standardized incidence rate (ASIR), age-standardized death rate (ASDR), and corresponding estimated annual percentage change (EAPC). RESULTS: The number of myocarditis incidence increased by 62.19%, from 780,410 cases in 1990 to 1,265,770 cases in 2019. The ASIR decreased by 4.42% (95%CI, from -0.26% to -0.21%) over the past 30 years. The number of deaths from myocarditis increased by 65.40% from 19,618 in 1990 to 324,490 in 2019, but the ASDR was relatively stable over the investigated period. ASDR increased in low-middle SDI regions (EAPC=0.48; 95%CI, 0.24 to 0.72) and decreased in low SDI regions (EAPC=-0.97; 95%CI, from -1.05 to -0.89). The age-standardized DALY rate decreased by 1.19% (95%CI, from -1.33% to -1.04%) per year. CONCLUSIONS: Globally, the ASIR and DALY for myocarditis decreased and the ASDR was stable over the past 30 years. The risk of incidences and death cases increased with age. Measures should be taken to control the risk of myocarditis in high-burden regions. Medical supplies should be improved in the high-middle SDI regions and middle SDI regions to reduce the deaths from myocarditis in these regions.
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Carga Global de Enfermedades , Miocarditis , Humanos , Años de Vida Ajustados por Calidad de Vida , Miocarditis/epidemiología , Salud Global , Años de Vida Ajustados por Discapacidad , IncidenciaRESUMEN
Single-stranded DNA-binding proteins (SSBs) play a crucial role in DNA metabolism by binding and stabilizing single-stranded DNA (ssDNA) intermediates. Through their multifaceted roles in DNA replication, recombination, repair, replication restart, and other cellular processes, SSB emerges as a central player in maintaining genomic integrity. These attributes collectively position SSBs as essential guardians of genomic integrity, establishing interactions with an array of distinct proteins. Unlike Escherichia coli, which contains only one type of SSB, some bacteria have two paralogous SSBs, referred to as SsbA and SsbB. In this study, we identified Staphylococcus aureus SsbA (SaSsbA) as a fresh addition to the roster of the anticancer drug 5-fluorouracil (5-FU) binding proteins, thereby expanding the ambit of the 5-FU interactome to encompass this DNA replication protein. To investigate the binding mode, we solved the complexed crystal structure with 5-FU at 2.3 Å (PDB ID 7YM1). The structure of glycerol-bound SaSsbA was also determined at 1.8 Å (PDB ID 8GW5). The interaction between 5-FU and SaSsbA was found to involve R18, P21, V52, F54, Q78, R80, E94, and V96. Based on the collective results from mutational and structural analyses, it became evident that SaSsbA's mode of binding with 5-FU diverges from that of SaSsbB. This complexed structure also holds the potential to furnish valuable comprehension regarding how 5-FU might bind to and impede analogous proteins in humans, particularly within cancer-related signaling pathways. Leveraging the information furnished by the glycerol and 5-FU binding sites, the complexed structures of SaSsbA bring to the forefront the potential viability of several interactive residues as potential targets for therapeutic interventions aimed at curtailing SaSsbA activity. Acknowledging the capacity of microbiota to influence the host's response to 5-FU, there emerges a pressing need for further research to revisit the roles that bacterial and human SSBs play in the realm of anticancer therapy.
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Antineoplásicos , Proteínas Bacterianas , Humanos , Proteínas Bacterianas/metabolismo , Glicerol , ADN de Cadena Simple , Fluorouracilo/farmacología , Escherichia coli/metabolismo , Replicación del ADN , Antineoplásicos/farmacología , Unión Proteica/genéticaRESUMEN
Allantoinase (ALLase; EC 3.5.2.5) possesses a binuclear metal center in which two metal ions are bridged by a posttranslationally carbamylated lysine. ALLase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of allantoin into allantoate. Biochemically, ALLase belongs to the cyclic amidohydrolase family, which also includes dihydropyrimidinase, dihydroorotase, hydantoinase (HYDase), and imidase. Previously, the crystal structure of ALLase from Escherichia coli K-12 (EcALLase-K12) was reported; however, the two active site loops crucial for substrate binding were not determined. This situation would limit further docking and protein engineering experiments. Here, we solved the crystal structure of E. coli BL21 ALLase (EcALLase-BL21) at a resolution of 2.07 Å (PDB ID 8HFD) to obtain more information for structural analyses. The structure has a classic TIM barrel fold. As compared with the previous work, the two missed active site loops in EcALLase-K12 were clearly determined in our structure of EcALLase-BL21. EcALLase-BL21 shared active site similarity with HYDase, an important biocatalyst for industrial production of semisynthetic penicillin and cephalosporins. Based on this structural comparison, we discussed the functional role of the two active site loops in EcALLase-BL21 to better understand the substrate/inhibitor binding mechanism for further biotechnological and pharmaceutical applications.
Asunto(s)
Escherichia coli K12 , Escherichia coli , Escherichia coli/metabolismo , Dominio Catalítico , Amidohidrolasas/química , Catálisis , Cristalografía por Rayos X , Sitios de UniónRESUMEN
BACKGROUND: Glycosyltransferases play a crucial role in various cancers. ß1, 3-N-acetylglucosaminyltransferase 2, a polylactosamine synthase, is an important member of the glycosyltransferase family. However, the biological function and regulatory mechanism of ß3GNT2 in esophageal carcinoma (ESCA) is still poorly understood. METHODS: The Cancer Genome Atlas and Genotype-Tissue Expression databases were used for gene expression and prognosis analysis. Quantitative real-time PCR, Western blot, and immunohistochemistry were performed to detect the expression of ß3GNT2 in ESCA cell lines and tissues. In vitro assays and xenograft tumor models were utilized to evaluate the impact of ß3GNT2 on ESCA progression. The downstream effectors and upstream regulators of ß3GNT2 were predicted by online software and verified by functional experiments. RESULTS: We found that ß3GNT2 was highly expressed in ESCA tissues and positively correlated with poor prognosis in ESCA patients. ß3GNT2 expression was closely associated with the tumor size, TNM stage, and overall survival of ESCA patients. Functionally, ß3GNT2 promoted ESCA cell growth, migration, and invasion in vitro, as well as tumorigenesis in vivo. Mechanistically, ß3GNT2 knockdown decreased the expression of the polylactosamine on EGFR. Knockdown of ß3GNT2 also inhibited the JAK/STAT signaling pathway. Meanwhile, the JAK/STAT inhibitor could partly reverse the biological effects caused by ß3GNT2 overexpression. Moreover, ß3GNT2 expression was positively regulated by CREB1 and negatively regulated by miR-133b. Both CREB1 and miR-133b was involved in the ß3GNT2-mediated ESCA progression. CONCLUSIONS: Our study, for the first time, reveals the importance of ß3GNT2 in ESCA progression and offers a potential therapeutic target for ESCA.
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Carcinoma , Neoplasias Esofágicas , MicroARNs , N-Acetilglucosaminiltransferasas/genética , Carcinoma/genética , Línea Celular Tumoral , Proliferación Celular , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Humanos , MicroARNs/genéticaRESUMEN
BACKGROUND: Co-trimoxazole, a sulfonamide antibiotic, is used to treat a variety of infections worldwide, and it remains a common first-line medicine for prophylaxis against Pneumocystis jiroveci pneumonia. However, it can cause severe cutaneous adverse reaction (SCAR), including Stevens-Johnson syndrome, toxic epidermal necrolysis, and drug reaction with eosinophilia and systemic symptoms. The pathomechanism of co-trimoxazole-induced SCAR remains unclear. OBJECTIVE: We aimed to investigate the genetic predisposition of co-trimoxazole-induced SCAR. METHODS: We conducted a multicountry case-control association study that included 151 patients with of co-trimoxazole-induced SCAR and 4631 population controls from Taiwan, Thailand, and Malaysia, as well as 138 tolerant controls from Taiwan. Whole-genome sequencing was performed for the patients and population controls from Taiwan; it further validated the results from Thailand and Malaysia. RESULTS: The whole-genome sequencing study (43 case patients vs 507 controls) discovered that the single-nucleotide polymorphism rs41554616, which is located between the HLA-B and MICA loci, had the strongest association with co-trimoxazole-induced SCAR (P = 8.2 × 10-9; odds ratio [OR] = 7.7). There were weak associations of variants in co-trimoxazole-related metabolizing enzymes (CYP2D6, GSTP1, GCLC, N-acetyltransferase [NAT2], and CYP2C8). A replication study using HLA genotyping revealed that HLA-B∗13:01 was strongly associated with co-trimoxazole-induced SCAR (the combined sample comprised 91 case patients vs 2545 controls [P = 7.2 × 10-21; OR = 8.7]). A strong HLA association was also observed in the case patients from Thailand (P = 3.2 × 10-5; OR = 3.6) and Malaysia (P = .002; OR = 12.8), respectively. A meta-analysis and phenotype stratification study further indicated a strong association between HLA-B∗13:01 and co-trimoxazole-induced drug reaction with eosinophilia and systemic symptoms (P = 4.2 × 10-23; OR = 40.1). CONCLUSION: This study identified HLA-B∗13:01 as an important genetic factor associated with co-trimoxazole-induced SCAR in Asians.
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Antibacterianos/efectos adversos , Antiinfecciosos Urinarios/efectos adversos , Pueblo Asiatico/genética , Hipersensibilidad a las Drogas/genética , Predisposición Genética a la Enfermedad , Antígenos HLA-B/genética , Combinación Trimetoprim y Sulfametoxazol/efectos adversos , Adolescente , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Humanos , Malasia/epidemiología , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Taiwán/epidemiología , Tailandia/epidemiología , Secuenciación Completa del Genoma , Adulto JovenRESUMEN
Single-stranded DNA (ssDNA)-binding protein (SSB) plays a crucial role in DNA replication, repair, and recombination as well as replication fork restarts. SSB is essential for cell survival and, thus, is an attractive target for potential antipathogen chemotherapy. Whether naturally occurring products can inhibit SSB remains unknown. In this study, the effect of the flavonols myricetin, quercetin, kaempferol, and galangin on the inhibition of Pseudomonas aeruginosa SSB (PaSSB) was investigated. Furthermore, SSB was identified as a novel quercetin-binding protein. Through an electrophoretic mobility shift analysis, myricetin could inhibit the ssDNA binding activity of PaSSB with an IC50 of 2.8 ± 0.4 µM. The effect of quercetin, kaempferol, and galangin was insignificant. To elucidate the flavonol inhibition specificity, the crystal structure of PaSSB complexed with the non-inhibitor quercetin was solved using the molecular replacement method at a resolution of 2.3 Å (PDB entry 7VUM) and compared with a structure with the inhibitor myricetin (PDB entry 5YUN). Although myricetin and quercetin bound PaSSB at a similar site, their binding poses were different. Compared with myricetin, the aromatic ring of quercetin shifted by a distance of 4.9 Å and an angle of 31o for hydrogen bonding to the side chain of Asn108 in PaSSB. In addition, myricetin occupied and interacted with the ssDNA binding sites Lys7 and Glu80 in PaSSB whereas quercetin did not. This result might explain why myricetin could, but quercetin could not, strongly inhibit PaSSB. This molecular evidence reveals the flavonol inhibition specificity and also extends the interactomes of the natural anticancer products myricetin and quercetin to include the OB-fold protein SSB.
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Proteínas de Unión al ADN/antagonistas & inhibidores , Flavonoles/farmacología , Pseudomonas aeruginosa/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Flavonoides/farmacología , Flavonoles/química , Quempferoles/farmacología , Modelos Moleculares , Conformación Proteica , Quercetina/química , Quercetina/farmacologíaRESUMEN
Single-stranded DNA (ssDNA)-binding proteins (SSBs) play a central role in cells by participating in DNA metabolism, including replication, repair, recombination, and replication fork restart. SSBs are essential for cell survival and thus an attractive target for potential anti-pathogen chemotherapy. In this study, we determined the crystal structure and examined the size of the ssDNA-binding site of an SSB from Salmonella enterica serovar Typhimurium LT2 (SeSSB), a ubiquitous opportunistic pathogen which is highly resistant to antibiotics. The crystal structure was solved at a resolution of 2.8 Å (PDB ID 7F25), indicating that the SeSSB monomer possesses an oligonucleotide/oligosaccharide-binding (OB) fold domain at its N-terminus and a flexible tail at its C-terminus. The core of the OB-fold in the SeSSB is made of a six-stranded ß-barrel capped by an α-helix. The crystal structure of the SeSSB contained two monomers per asymmetric unit, which may indicate the formation of a dimer. However, the gel-filtration chromatography analysis showed that the SeSSB forms a tetramer in solution. Through an electrophoretic mobility shift analysis, we characterized the stoichiometry of the SeSSB complexed with a series of ssDNA dA homopolymers, and the size of the ssDNA-binding site was determined to be around 22 nt. We also found the flavanonol taxifolin, also known as dihydroquercetin, capable of inhibiting the ssDNA-binding activity of the SeSSB. Thus, this result extended the SSB interactome to include taxifolin, a natural product with a wide range of promising pharmacological activities.
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Salmonella enterica , ADN de Cadena Simple , Proteínas de Unión al ADN/metabolismo , Unión Proteica , Quercetina/análogos & derivados , Quercetina/farmacología , Salmonella enterica/genética , Salmonella typhimurium/genéticaRESUMEN
Interest in the third-row transition metal osmium and its compounds as potential anticancer agents has grown in recent years. Here, we synthesized the osmium(VI) nitrido complex Na[OsVI(N)(tpm)2] (tpm = [5-(Thien-2-yl)-1H-pyrazol-3-yl]methanol), which exhibited a greater inhibitory effect on the cell viabilities of the cervical, ovarian, and breast cancer cell lines compared with cisplatin. Proteomics analysis revealed that Na[OsVI(N)(tpm)2] modulates the expression of protein-transportation-associated, DNA-metabolism-associated, and oxidative-stress-associated proteins in HepG2 cells. Perturbation of protein expression activity by the complex in cancer cells affects the functions of the mitochondria, resulting in high levels of cellular oxidative stress and low rates of cell survival. Moreover, it caused G2/M phase cell cycle arrest and caspase-mediated apoptosis of HepG2 cells. This study reveals a new high-valent osmium complex as an anticancer agent candidate modulating protein homeostasis.
Asunto(s)
Antineoplásicos , Osmio , Humanos , Osmio/farmacología , Células Hep G2 , Proteostasis , Antineoplásicos/farmacología , Apoptosis , Línea Celular TumoralRESUMEN
Single-stranded DNA-binding proteins (SSBs) are essential to cells because they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. Some bacteria possess more than one paralogous SSB. Three similar SSBs, namely, SsbA, SsbB, and SsbC, are found in Staphylococcus aureus. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to SSBs remains unknown. In this study, we found that 5-FU could form a stable complex with S. aureus SsbB (SaSsbB). We cocrystallized 5-FU with SaSsbB and solved complex structures to assess binding modes. Two complex forms of the structures were determined, namely, the individual asymmetric unit (two SaSsbB monomers) containing one (PDB entry 7D8J) or two 5-FU molecules (PDB entry 7DEP). The locations of 5-FU in these two SaSsbB complexes were similar regardless of the binding ratio. The structures revealed that residues T12, K13, T30, F48, and N50 of SaSsbB were involved in 5-FU binding. The mutations of T12, K13, and F48 caused the low 5-FU binding activity of SaSsbB, a result consistent with the structural analysis results. Taken together, the complexed structure and the binding mode analysis of SaSsbB extended the anticancer drug 5-FU interactome to include the oligonucleotide/oligosaccharide-binding fold protein.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Fluorouracilo/química , Antimetabolitos Antineoplásicos/química , Antimetabolitos Antineoplásicos/metabolismo , Proteínas Bacterianas/genética , Cristalización , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Fluorouracilo/metabolismo , Mutagénesis Sitio-Dirigida , Staphylococcus aureus/químicaRESUMEN
Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides and considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to DHOase remains unknown. Here, we report the crystal structures of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with malate, 5-FU, and 5-aminouracil (5-AU). ScDHOase shares structural similarity with Escherichia coli DHOase. We also characterized the binding of 5-FU and 5-AU to ScDHOase by using the fluorescence quenching method. These complexed structures revealed that residues Arg18, Asn43, Thr106, and Ala275 of ScDHOase were involved in the 5-FU (PDB entry 6L0B) and 5-AU binding (PDB entry 6L0F). Overall, these results provide structural insights that may facilitate the development of new inhibitors targeting DHOase and constitute the 5-FU and 5-AU interactomes for further clinical chemotherapies.
Asunto(s)
Antineoplásicos/química , Dihidroorotasa/química , Fluorouracilo/química , Saccharomyces cerevisiae/enzimología , Uracilo/análogos & derivados , Antineoplásicos/farmacología , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Dihidroorotasa/metabolismo , Escherichia coli/enzimología , Fluorouracilo/farmacología , Malatos/química , Modelos Moleculares , Unión Proteica , Uracilo/química , Uracilo/farmacologíaRESUMEN
PriB is a primosomal protein required for the replication fork restart in bacteria. Although PriB shares structural similarity with SSB, they bind ssDNA differently. SSB consists of an N-terminal ssDNA-binding/oligomerization domain (SSBn) and a flexible C-terminal protein-protein interaction domain (SSBc). Apparently, the largest difference in structure between PriB and SSB is the lack of SSBc in PriB. In this study, we produced the chimeric PriB-SSBc protein in which Klebsiella pneumoniae PriB (KpPriB) was fused with SSBc of K. pneumoniae SSB (KpSSB) to characterize the possible SSBc effects on PriB function. The crystal structure of KpSSB was solved at a resolution of 2.3 Å (PDB entry 7F2N) and revealed a novel 114-GGRQ-117 motif in SSBc that pre-occupies and interacts with the ssDNA-binding sites (Asn14, Lys74, and Gln77) in SSBn. As compared with the ssDNA-binding properties of KpPriB, KpSSB, and PriB-SSBc, we observed that SSBc could significantly enhance the ssDNA-binding affinity of PriB, change the binding behavior, and further stimulate the PriA activity (an initiator protein in the pre-primosomal step of DNA replication), but not the oligomerization state, of PriB. Based on these experimental results, we discuss reasons why the properties of PriB can be retrofitted when fusing with SSBc.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Klebsiella pneumoniae/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Replicación del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Homología de SecuenciaRESUMEN
Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.