Transcription Factor P53 Research Articles

Effects of Malondialdehyde on Growth Performance, Gastrointestinal Health, and Muscle Quality of Striped Catfish (Pangasianodon hypophthalmus)

Malondialdehyde (MDA) is a reactive carbonyl compound produced through lipid peroxidation during feed storage, which poses a significant threat to fish health. This study aimed to evaluate the effects of dietary MDA on the growth rate, gastrointestinal health, and muscle quality of striped catfish (Pangasianodon hypophthalmus). A basal diet (M0) containing 34% crude protein and 10.5% crude lipid was formulated. Each group was sprayed with malondialdehyde solution (0, 5, 10, 20, 40, and 80 mg/kg, on dietary crude lipid basis; 0, 0.53, 1.07, 2.13, 4.26, and 8.52 mg/kg, on dietary basis) before feeding, respectively. Each diet was randomly assigned to triplicates of 30 striped catfish (initial weight 31.38 g) per net cage. After 8 weeks, dietary inclusion of MDA regardless of level significantly depressed the growth rate and feed utilization. The extent of structural damage to the gastrointestinal tract increased progressively with increasing dietary MDA levels. The extent of damage to the intestinal biological barrier (intestinal microbial structure), chemical barrier (trypsin, lipase, amylase, and maltase activity), physical barrier (zonula occludent-2, occludin, claudin 7α, and claudin 12 relative expression), and immune barrier (contents of complement 4, complement 3, immunoglobulin M, and lysozyme activity) was dose-related to dietary MDA. Moreover, a linear decline in the activities of intestinal antioxidant enzymes (catalas, superoxide dismutase, et al.) and anti-inflammatory factor (transforming growth factor beta1, interleukin 10) relative expression was noted alongside an increase in dietary MDA content. In contrast, the relative expression levels of intestinal inflammatory factor (interleukin 8, transcription factor p65, tumor necrosis factor alpha) relative expression displayed an opposing trend. Additionally, dietary MDA exerted a linear influence on muscle color and texture characteristics. In conclusion, high doses of MDA (5–80 mg/kg) reduced the growth performance of striped catfish, attributed to linear damage to the gastrointestinal tract, a linear decrease in antioxidant function, and the occurrence of an inflammatory response. High doses of MDA (>40 mg/kg) were observed to significantly increase dorsal muscle b-value and induce muscle yellowing.

REGγ-mediated Barrier Disruption and NF-κB Activation Aggravates Intestinal Inflammation in Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) stands as the most prevalent and severe gastrointestinal ailment affecting premature newborns, particularly those with extremely low birth weights. Intestinal barrier dysfunction emerges as a crucial factor in NEC's pathogenesis. REGγ predominantly manifests in the intestinal epithelium and has shown to regulate experimental colitis. However, the potential correlation between REGγ and NEC remains ambiguous. This study reveals that REGγ is notably overexpressed in both human and murine NEC samples. REGγ-deficient mice displayed improvements in intestinal mucosal inflammation and reduced NEC severity. Additionally, REGγ deficiency notably lowered the expression of phosphorylated transcription factor p65 and inflammatory factors in intestinal epithelial cells of NEC-afflicted mice. REGγ exacerbates the local inflammation by triggering the degradation of IκBɛ, a negative regulator of NFκB. Clinically, REGγ and p65 expression levels exhibit a positive correlation in NEC specimens. Moreover, pre-treatment of mice with a p65 inhibitor effectively suppressed the expression of inflammatory mediators and alleviated REGγ-mediated NEC development. These findings underscore that abnormal upregulation of REGγ triggers NEC development by enhancing NF-κB activity and exacerbating epithelial barrier dysfunction. Thus, novel therapeutic approaches targeting REGγ inhibition may offer enhanced treatment for NEC.

BRD4 sustains p63 transcriptional program in keratinocytes

Here, we investigated the potential interaction between bromodomain-containing protein 4 (BRD4), an established epigenetic modulator and transcriptional coactivator, and p63, a member of the p53 transcription factor family, essential for epithelial development and skin homeostasis. Our protein–protein interaction assays demonstrated a strong and conserved physical interaction between BRD4 and the p53 family members—p63, p73, and p53—suggesting a shared binding region among these proteins. While the role of BRD4 in cancer development through its interaction with p53 has been explored, the effects of BRD4 and Bromodomain and Extra Terminal (BET) inhibitors in non-transformed cells, such as keratinocytes, remain largely unknown. Our functional analyses revealed changes in cellular proliferation and differentiation in keratinocytes depleted of either p63 or BRD4, which were further supported by using the BRD4 inhibitor JQ1. Transcriptomic analyses, chromatin immunoprecipitation, and RT-qPCR indicated a synergistic mechanism between p63 and BRD4 in regulating the transcription of keratinocyte-specific p63 target genes, including HK2, FOXM1, and EVPL. This study not only highlights the complex relationship between BRD4 and p53 family members but also suggests a role for BRD4 in maintaining keratinocyte functions. Our findings pave the way for further exploration of potential therapeutic applications of BRD4 inhibitors in treating skin disorders.

Crosstalk between paralogs and isoforms influences p63-dependent regulatory element activity.

The p53 family of transcription factors (p53, p63 and p73) regulate diverse organismal processes including tumor suppression, maintenance of genome integrity and the development of skin and limbs. Crosstalk between transcription factors with highly similar DNA binding profiles, like those in the p53 family, can dramatically alter gene regulation. While p53 is primarily associated with transcriptional activation, p63 mediates both activation and repression. The specific mechanisms controlling p63-dependent gene regulatory activity are not well understood. Here, we use massively parallel reporter assays (MPRA) to investigate how local DNA sequence context influences p63-dependent transcriptional activity. Most regulatory elements with a p63 response element motif (p63RE) activate transcription, although binding of the p63 paralog, p53, drives a substantial proportion of that activity. p63RE sequence content and co-enrichment with other known activating and repressing transcription factors, including lineage-specific factors, correlates with differential p63RE-mediated activities. p63 isoforms dramatically alter transcriptional behavior, primarily shifting inactive regulatory elements towards high p63-dependent activity. Our analysis provides novel insight into how local sequence and cellular context influences p63-dependent behaviors and highlights the key, yet still understudied, role of transcription factor paralogs and isoforms in controlling gene regulatory element activity.

Role of apoptosis-associated proteins p53 and bcl-2 in the pathogenesis of nervous system diseases

Diseases of the central nervous system occupy a leading place, along with cardiovascular and oncological diseases, and the proportion of patients suffering from diseases of the nervous system is increasing as the population ages. This group of diseases includes acute conditions, such as ischemic stroke, and chronic multifactorial diseases — Alzheimer's and Parkinson's diseases, epilepsy, etc. The development of specific methods for their treatment is difficult, and these drugs are not very effective. Almost all brain diseases are based on common mechanisms such as oxidative stress, inflammation and neuronal death. Most often, cells die by apoptosis due to an imbalance between pro-apoptotic and anti-apoptotic factors. This work examines two of them: the apoptosis-promoting transcription factor and tumor suppressor p53 and its opposing B-cell lymphoma protein Bcl-2. The choice of these proteins for study is due to the fact that both proteins are key regulators of apoptosis and are important in the pathogenesis of nervous diseases, since neurons are not highly proliferating cells. The p53 protein is involved in the regulation of many genes responsible for DNA repair, apoptosis, and other biochemical cellular processes; this is especially important when studying neuronal pathology. Bcl-2 suppresses apoptosis in various cells, including neurons, by controlling mitochondrial membrane permeability and inhibiting caspases. In diseases, its expression can either increase, for example, in the case of malignant tumors, or decrease, as in the case of neurodegenerative processes. It has been established that p53 and Bcl-2 are in close interaction in the process of regulating apoptosis; their ratio may be an important prognostic factor. The purpose of this work was to assess the role of these proteins in the pathogenesis of various diseases of the nervous system, and to search for general patterns of changes in their expression and coexpression.

Deciphering the molecular mechanism underlying morphology transition in two-component DNA-protein cophase separation.

Nucleic acid and protein co-condensates exhibit diverse morphologies crucial for fundamental cellular processes. Despite many previous studies that advanced our understanding of this topic, several interesting biophysical questions regarding the underlying molecular mechanisms remain. We investigated DNA and human transcription factor p53 co-condensates-a scenario where neither dsDNA nor the protein demonstrates phase-separation behavior individually. Through a combination of experimental assays and theoretical approaches, we elucidated: (1) the phase diagram of DNA-protein co-condensates at a certain observation time, identifying a phase transition between viscoelastic fluid and viscoelastic solid states, and a morphology transition from droplet-like to "pearl chain"-like co-condensates; (2) the growth dynamics of co-condensates. Droplet-like and "pearl chain"-like co-condensates share a common initial critical microscopic cluster size at the nanometer scale during the early stage of phase separation. These findings provide important insights into the biophysical mechanisms underlying multi-component phase separation within cellular environments.

Genome-Wide Analysis of p53 Targets Reveals SCN2A as a Novel Player in p53-Induced Cell Arrest in HPV-Positive Cells.

The host transcription factor p53 is a critical tumor suppressor in HPV-induced carcinogenesis, regulating target genes involved in cell cycle arrest and apoptosis. However, the p53 targets have not been thoroughly analyzed in HPV-infected cells. In this study, p53 signaling in HPV16 and HPV18 cells was activated by depleting the viral oncoprotein E6. Subsequently, p53-regulated genes were identified by comparing them with genes altered in p53-silenced cells. True p53 targets were defined as genes with at least one overlapping p53 binding site and ChIP peak near their locus. Our analysis revealed that while some p53 targets were common to both the HPV16 and HPV18 cells, the majority of the targets differed between these two types, potentially contributing to the varying prevalence of HPV16 and HPV18 in cervical cancer. Additionally, we identified SCN2A as a novel p53 target involved in p53-induced cell cycle arrest in HPV-related carcinogenesis. This study provides new insights into the mechanisms by which p53 inhibits HPV-induced carcinogenesis.

Temporal regulation of gene expression through integration of p53 dynamics and modifications.

The master regulator of the DNA damage response, the transcription factor p53, orchestrates multiple downstream responses and coordinates repair processes. In response to double-strand DNA breaks, p53 exhibits pulses of expression, but how it achieves temporal coordination of downstream responses remains unclear. Here, we show that p53's posttranslational modification state is altered between its first and second pulses of expression. We show that acetylations at two sites, K373 and K382, were reduced in the second pulse, and these acetylations differentially affected p53 target genes, resulting in changes in gene expression programs over time. This interplay between dynamics and modification may offer a strategy for cellular hubs like p53 to temporally organize multiple processes in individual cells.

TP53: the unluckiest of genes?

The transcription factor p53 plays a key role in the cellular defense against cancer development. It is inactivated in virtually every tumor, and in every second tumor this inactivation is due to a mutation in the TP53 gene. In this perspective, we show that this diverse mutational spectrum is unique among all other cancer-associated proteins and discuss what drives the selection of TP53 mutations in cancer. We highlight that several factors conspire to make the p53 protein particularly vulnerable to inactivation by the mutations that constantly plague our genome. It appears that the TP53 gene has emerged as a victim of its own evolutionary past that shaped its structure and function towards a pluripotent tumor suppressor, but came with an increased structural fragility of its DNA-binding domain. TP53 loss of function - with associated dominant-negative effects - is the main mechanism that will impair TP53 tumor suppressive function, regardless of whether a neomorphic phenotype is associated with some of these variants.

Co-Regulation Mechanism of Host p53 and Fos in Transcriptional Activation of ILTV Immediate-Early Gene ICP4.

Infectious laryngotracheitis virus (ILTV) exhibits a cascade expression pattern of encoded genes, and ICP4 is the only immediate-early gene of ILTV, which plays a crucial role in initiating the subsequent viral genes. Therefore, studying the transcriptional regulation mechanism of ICP4 holds promise for effectively blocking ILTV infection and spread. Host transcriptional factors p53 and Fos are proven to regulate a variety of viral infections, and our previous studies have demonstrated their synergistic effects in regulating ILTV infection. In this study, we constructed eukaryotic expression vectors for p53 and Fos as well as their specific siRNAs and transfected them into a chicken hepatoma cell line. The results showed that knocking down p53 or Fos significantly inhibited ICP4 transcription, while overexpressing p53 or Fos had an opposite effect. A further CoIP and ChIP-qPCR assay suggested p53 and Fos physically interacted with each other, and jointly bound to the upstream transcriptional regulatory region of ICP4. To elucidate the specific mechanisms of p53 and Fos in regulating ICP4 transcription, we designed p53 and Fos protein mutants by mutating their DNA binding domains, which significantly reduced their binding ability to DNA without affecting their interaction. The results showed that Fos directly bound to the promoter region of ICP4 as a binding target of p53, and the p53-Fos protein complex acted as a transcriptional co-regulator of ICP4. Studying the transcriptional process and regulatory pattern of ICP4 is of great significance for understanding the molecular mechanism of ILTV infection, and thus for finding effective methods to control and prevent it.

Epigenetic regulation of p63 blocks squamous-to-neuroendocrine transdifferentiation in esophageal development and malignancy.

While cell fate determination and maintenance are important in establishing and preserving tissue identity and function during development, aberrant cell fate transition leads to cancer cell heterogeneity and resistance to treatment. Here, we report an unexpected role for the transcription factor p63 (Trp63/TP63) in the fate choice of the squamous versus neuroendocrine lineage in esophageal development and malignancy. Deletion of p63 results in extensive neuroendocrine differentiation in the developing mouse esophagus and esophageal progenitors derived from human embryonic stem cells. In human esophageal neuroendocrine carcinoma (eNEC) cells, p63 is transcriptionally silenced by EZH2-mediated H3K27 trimethylation (H3K27me3). Up-regulation of the major p63 isoform ΔNp63α, through either ectopic expression or EZH2 inhibition, promotes squamous transdifferentiation of eNEC cells. Together, these findings uncover p63 as a rheostat in coordinating the transition between squamous and neuroendocrine cell fates during esophageal development and tumor progression.

Cultures derived from pancreatic cancer xenografts with long-term gemcitabine treatment produce chemoresistant secondary xenografts: Establishment of isogenic gemcitabine-sensitive and -resistant models

In attempts to establish sophisticated models to reproduce the process of acquired drug resistance, we transformed normal human pancreatic ductal epithelial cells by introducing genes for multiple cellular factors. We also created isogenic gemcitabine-sensitive and -resistant models by short- and long-term gemcitabine treatment, respectively. These models demonstrated differences in drug resistance in vivo, but not in vitro. Gemcitabine treatment also induced squamous transdifferentiation in xenografts in mice. The transcription factor p63 was identified as a possible resistance-determining factor but was unlikely to be solely responsible for the resistance to gemcitabine. This system would prove useful to discover novel molecular targets to overcome chemotherapy resistance, by allowing the evaluation of molecules of interest in xenograft models after in vitro genetic ablation.

P53motifDB: integration of genomic information and tumor suppressor p53 binding motifs.

The tumor suppressor gene TP53 encodes the DNA binding transcription factor p53 and is one of the most commonly mutated genes in human cancer. Tumor suppressor activity requires binding of p53 to its DNA response elements and subsequent transcriptional activation of a diverse set of target genes. Despite decades of close study, the logic underlying p53 interactions with its numerous potential genomic binding sites and target genes is not yet fully understood. Here, we present a database of DNA and chromatin-based information focused on putative p53 binding sites in the human genome to allow users to generate and test new hypotheses related to p53 activity in the genome. Users can query genomic locations based on experimentally observed p53 binding, regulatory element activity, genetic variation, evolutionary conservation, chromatin modification state, and chromatin structure. We present multiple use cases demonstrating the utility of this database for generating novel biological hypotheses, such as chromatin-based determinants of p53 binding and potential cell type-specific p53 activity. All database information is also available as a precompiled sqlite database for use in local analysis or as a Shiny web application.

Thermostable Nucleoid Protein Cren7 Slides Along DNA and Rapidly Dissociates From DNA While Not Inhibiting the Sliding of Other DNA-binding Protein

A nucleoid protein Cren7 compacts DNA, contributing to the living of Crenarchaeum in high temperature environment. In this study, we investigated the dynamic behavior of Cren7 on DNA and its functional relation using single-molecule fluorescence microscopy. We found two mobility modes of Cren7, sliding along DNA and pausing on it, and the rapid dissociation kinetics from DNA. The salt dependence analysis suggests a sliding with continuous contact to DNA, rather than hopping/jumping. The mutational analysis demonstrates that Cren7 slides along DNA while Trp (W26) residue interacts with the DNA. Furthermore, Cren7 does not impede the target search by a model transcription factor p53, implying no significant interference to other DNA-binding proteins on DNA. At high concentration of Cren7, the molecules form large clusters on DNA via bridging, which compacts DNA. We discuss how the dynamic behavior of Cren7 on DNA enables DNA-compaction and protein-bypass functions.

Cytotoxicity of Activator Expression in CRISPR-based Transcriptional Activation Systems.

CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of published and new CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system. Based on our findings for the SAM system, we extended our studies to additional ADs and the p300 acetyltransferase core domain. We show that the expression of potent transcriptional activators in lentiviral producer cells leads to low lentiviral titers, while their expression in the transduced target cells leads to cell death. Using inducible lentiviral vectors, we could not identify an activator expression window for effective SAM-based CRISPRa without measurable toxicity. The toxicity of current SAM-based CRISPRa systems hinders their wide adoption in biomedical research and introduces selection bottlenecks that may confound genetic screens. Our results suggest that the further development of CRISPRa technology should consider both the efficiency of gene activation and activator toxicity.

Abstract B084: Comparative functional analysis of TP53 alleles in pancreatic ductal adenocarcinoma in vivo

Abstract TP53 is the second most mutated gene in pancreatic ductal adenocarcinoma (PDAC), bearing both truncal loss-of-function and missense point mutations. TP53 encodes a stress-induced transcription factor (p53) capable of activating tumor suppressive pathways mediating cell cycle arrest, apoptosis, senescence, and metabolic reprogramming. Despite high mutational frequency, how TP53 mutants function to suppress pancreatic tumorigenesis in vivo remains unclear. Previous studies indicate that the most frequent point mutations observed in human PDAC (TP53 R175H and R273H; mouse homologues Trp53 R172H and R270H) exhibit both dominant-negative (e.g., response to radiation in leukemia cells) and gain-of-function (e.g., increased liver metastasis in advanced PDAC models) roles compared to p53 null cells in various contexts. To rigorously explore the differential functions of p53 point and deletion mutants in PDAC progression, we performed an isogenic comparative analysis of Trp53 alleles in vivo using Mosaic Analysis with Double Markers (MADM) in mice. MADM uses stochastic mitotic recombination to induce two genotypically distinct daughter cells – created at 1-to-1 ratios – expressing different Trp53 variants dependent on the genotype of the mouse (+/+ vs. -/-, R172H/R172H vs. +/+, R172H/R172H vs. -/-, R270H/R270H vs. +/+, R270H/R270H vs. -/-) and simultaneously label them with unique genetically encoded fluorescent markers (TdTomato vs. GFP). We integrated MADM into a faithful Kras-driven model of pancreatic tumorigenesis (KC: Kras LSL-G12D/+ ; Pdx1-Cre) and used fluorescence microscopy to trace subclonal populations with various Trp53 alleles within the same mouse. By analyzing the ratio of TdTomato+ to GFP+ cells, we studied the functional differences between alleles in driving tumor initiation and expansion at various stages of progression. Strikingly, we found that Trp53 -/- cells exhibited greater expansion in preinvasive pancreatic intraepithelial neoplasias (PanINs) compared to not only Trp53 +/+ cells but also cells harboring either point mutation (Trp53 R172H/R172H or Trp53 R270H/R270H ). While Trp53 R270H/R270H cells predominated over Trp53 +/+ in PanINs, Trp53 R172H/R172H cells were surprisingly indistinguishable from Trp53 +/+ cells, suggesting that p53R172H retains tumor suppressive properties in early PDAC progression. Conversely, both Trp53 R172H/R172H and Trp53 R270H/R270H cells progressed to advanced PDAC with comparable PDAC incidence and survival as Trp53 -/- , arguing that Trp53 R172H is a separation-of-function mutant concordant with a distinct transcriptional profile relative to Trp53 -/- tumor cells. Together, these studies offer the strongest evidence to date for stage-specific functional differences between TP53 mutant variants in cancer progression in vivo and provide a faithful MADM-based PDAC model to define molecular mechanisms (through ongoing transcriptomic, epigenomic, and metabolomic studies) that govern functional differences amongst these diverse alleles. Citation Format: Sherry S. Agabiti, Andy Tang, Timothy Nottoli, Mandar D. Muzumdar. Comparative functional analysis of TP53 alleles in pancreatic ductal adenocarcinoma in vivo [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B084.

Genetic Mechanisms of Oral Leukoplakia: A Systematic Review

Objectives: Oral leukoplakia (OL) is the most common type of oral potentially malignant disorder. It is currently managed through lesion removal either by laser excision or resection. However, its multifactorial aetiology often results in recurrence and cancer transformation. Therefore, findings on novel biomarkers are emerging to understand OL formation and progression towards malignancy. We performed a systematic review to identify the genetic factors for the risk of OL conditions. Methods: The study protocol was registered in PROSPERO, ID: CRD42024497161. We searched PubMed, MEDLINE, Scopus, ScienceDirect, Web of Science, ClinicalKey and Wiley databases from 2018 to 2023. The study was conducted following PRISMA guidelines and articles were selected based on predefined inclusion criteria. Genomic profiles of OL tissues collected from study patients were summarised based on outcome determinants of predictive or diagnostic markers, in relation to OL histopathological features. Results: Seventeen studies met the inclusion criteria. Results showed that OL formation and progression involve genetic factors such as cytokines, proliferation antigen, major histocompatibility complex and CD molecules of lymphocytes, caspase, immune checkpoint, oral cancer key genes, nuclear proteins and transcription factors, toll-like receptor, macrophage and polycomb complex. Among them, the transcription factor p53 is the most investigated factor. However, the cytokine was found to play critical roles in OL progression towards the advanced stages, and is closely associated with dysplastic changes. Conclusion: Exposure to genetic alterations results in OL malignant transformation. Future studies on differential cytokines profiling of differentiated dysplasia, may reveal the novel stage-wise biomarkers for molecular pathological grading.

Radiosensitizing effects of CDK4/6 inhibitors in hormone receptor-positive and HER2-negative breast cancer mediated downregulation of DNA repair mechanism and NF-κB-signaling pathway

CDK4/6 inhibitors combined with endocrine therapy prolonged survival in hormone receptor (HR)-positive and HER2-negative advanced breast cancer. We investigated whether CDK4/6 inhibitors enhance radiosensitivity and their underlying mechanisms of this subtype of breast cancer. In vitro and in vivo experiments were conducted using two HR-positive and HER2-negative breast cancer cell lines (MCF-7 and T-47D), CDK4/6 inhibitors (ribociclib and palbociclib) and radiotherapy (RT) to assess the biological functions and mechanisms. The radiation-enhancing effect was assessed using clonogenic assays; γH2AX and 53BP1 levels were assessed by immunofluorescence to evaluate DNA damage. The levels of phospho (p)-ERK, c-Myc, and DNA-double strand break (DSB)-related molecules, p-DNA-PKcs, Rad51, and p-ATM, were assessed by western blotting. We used an NF-κB p65 transcription factor assay kit to evaluate NF-κB activity. We evaluated the antitumor effect of the combination of RT and ribociclib through the MCF-7 orthotopic xenograft model. The synergistic effects of combining RT with ribociclib and palbociclib pretreatment were demonstrated by clonogenic assay. CDK4/6 inhibitors synergistically increased the numbers of RT-induced γH2AX and 53BP1, downregulated the expression of p-DNA-PKcs, Rad51 and p-ATM activated by RT, and reduced RT-triggering p-ERK expression, NF-κB activation, and its down-streaming gene, c-Myc. Combined ribociclib and RT reduced the growth of MCF-7 cell xenograft tumors, and downregulated the immunohistochemical expression of p-ERK, p-NF-κB p65, and c-Myc compared to that in the control group. Combining CDK4/6 inhibitors enhanced radiosensitivity of HR-positive and HER2-negative breast cancer cells at least by reducing DNA-DSB repair and weakening the activation of ERK and NF-κB signaling by RT.

Deciphering the Mystery in p300 Taz2-p53 TAD2 Recognition.

Intrinsically disordered proteins (IDPs) engage in various fundamental biological activities, and their behavior is of particular importance for a better understanding of the verbose but well-organized signal transduction in cells. IDPs exhibit uniquely paradoxical features with low affinity but simultaneously high specificity in recognizing their binding targets. The transcription factor p53 plays a crucial role in cancer suppression, carrying out some of its biological functions using its disordered regions, such as N-terminal transactivation domain 2 (TAD2). Exploration of the binding and unbinding processes between proteins is challenging, and the inherently disordered properties of these regions further complicate the issue. Computer simulations are a powerful tool to complement the experiments to fill gaps to explore the binding/unbinding processes between proteins. Here, we investigated the binding mechanism between p300 Taz2 and p53 TAD2 through extensive molecular dynamics (MD) simulations using the physics-based UNited RESidue (UNRES) force field with additional Go̅-like potentials. Distance restraints extracted from the NMR-resolved structures were imposed on intermolecular residue pairs to accelerate binding simulations, in which Taz2 was immobilized in a native-like conformation and disordered TAD2 was fully free. Starting from six structures with TAD2 placed at different positions around Taz2, we observed a metastable intermediate state in which the middle helical segment of TAD2 is anchored in the binding pocket, highlighting the significance of the TAD2 helix in directing protein recognition. Physics-based binding simulations show that successful binding is achieved after a series of stages, including (1) protein collisions to initiate the formation of encounter complexes, (2) partial attachment of TAD2, and finally (3) full attachment of TAD2 to the correct binding pocket of Taz2. Furthermore, machine-learning-based PathDetect-SOM was used to identify two binding pathways, the encounter complexes, and the intermediate states.

Clinical Efficacy of Vaccaria segetalis Seeds and Gleditsia sinensis Lam Thorns on Prostate Cancer: A Preliminary Mechanism Analysis based on Network Pharmacology

Objective: The mechanism of Vaccaria segetalis (VS) seeds and Gleditsia sinensis Lam (GS) thorns in the treatment of prostate cancer (PC) was analyzed via network pharmacological analysis methods and molecular docking. Methods: The Traditional Chinese Medicine Systems Pharmacology Database Platform (TCMSP) was used to screen the PC’s effective components and targets; GeneCards and OMIM databases to search for targets related to PC. The intersection target was uploaded to the STRING database to obtain a proteinprotein interaction (PPI) network; and the key targets were screened from the PPI network via R language, CytoNCA, and CytoHubba tools. Gene Ontology (GO) and Kyoto encyclopedia of genes and genome (KEGG) pathway enrichment tools were used to analyze biological processes and molecular docking of key targets via AutoDock Vina software. Results: A total of 13 compounds, 229 nodes, 879 edges, and 20 key targets were obtained through the PPI network. Go and KEGG analysis showed that the intersection targets of VS and GS with PC were mainly involved in regulating cell promotion, cell apoptosis, cell cycle, and reversing epithelialmesenchymal transition (EMT) processing. Molecular docking revealed that the relevant targets of potential PC were characterized with stabilized affinity. Specifically, the targets with better affinity included estrogen receptor 1 (ESR1) with kaempferol, transcription factor p65 (RELA) with fisetin, kaempferol, quercetin, and mitogen-activated protein kinase 1 (MAPK1) with fisetin, and G1/S-specific cyclin-D1 (CCND1) with fisetin, kaempferol, and quercetin. Conclusion: In summary, this study reveals potential molecular therapeutic mechanisms of VS and GS in PC and provides a reference for the wide application of VS and GS in the clinical management of PC.