Search Results (173)

Background: Viral attachment mediated by host cell surface receptors is the first step in viral infection. As a key cell surface receptor, heparan sulfate (HS) mediates the attachment and entry of numerous non-enveloped viruses in livestock, thereby serving as a crucial molecular target for studying virus–host interactions. Methods: Based on the structural scaffold of a nanobody (Nb; PDB: 7TJC), we rationally designed and constructed a mutant Nb targeting HS, designated HS-Mut-Nb1, using molecular docking, site-directed mutagenesis, molecular dynamics (MD) simulations, and experimental characterization. Results: Molecular docking indicated that the active site of wild-type Nb for HS binding was located within the cavity jointly formed by the complementarity-determining region 3 (CDR3) and the framework regions (FRs) of the wild-type Nb. A comprehensive analysis integrating virtual alanine scanning, site-directed mutagenesis, and MD simulations revealed that the combination of three point mutations (Phe47Arg, Asp99Tyr, and Tyr108Pro) significantly enhanced the binding affinity of Mut-Nb1 for HS, with a calculated binding free energy (ΔG) of −83.26 ± 3.06 kcal/mol. Enzyme-linked immunosorbent assay (ELISA) results further confirmed that Mut-Nb1 exhibited high affinity for HS (K_D = 65.87 nM) and specificity (positive/negative ratio, P/N = 3.84; cross-reactivity, CR < 6.60%). Conclusions: This study not only provides novel candidate molecules for elucidating the mechanism of HS–virus interactions and developing related inhibitors but also offers a reference for the rapid construction of mutant Nbs. Full article

Reparative macrophage polarization and macrophage-derived reactive oxygen species (ROS) are required for ischemia-induced revascularization in peripheral artery disease (PAD). Our previous study showed that mitochondrial fission protein dynamin-related protein 1 (DRP1) promotes reparative polarization and metabolic reprogramming in macrophages and post-ischemic neovascularization. However, the redox-dependent mechanism governing DRP1 activation in this context remains elusive. Here, using a mouse hindlimb ischemia (HLI) model of PAD, we identify cysteine sulfenylation (CysOH) of DRP1 as a critical redox modification induced in ischemic bone marrow (BM)-derived cells. BM chimeric mice reconstituted with CRISPR/Cas9-generated “redox-dead” DRP1-C631A knock-in mutant (Drp1^C/A) BM exhibited markedly reduced limb perfusion recovery and CD31⁺ capillary density in ischemic muscles following HLI. These defects were associated with enhanced Ly6G⁺ neutrophil accumulation, pro-inflammatory F4/80⁺CD80⁺ M1-like macrophages and reduced anti-inflammatory F4/80⁺CD206⁺ M2-like macrophages in ischemic muscle. Mechanistically, using an in vitro PAD model, hypoxia serum starvation (HSS) rapidly induced NADPH oxidase 2-dependent cytosolic ROS production and DRP1-CysOH formation in wild-type macrophages. In contrast, Drp1^C/A macrophages failed to undergo DRP1-CysOH-dependent mitochondrial fission under HSS, resulting in aberrant metabolic reprogramming characterized by enhanced glycolysis and mitochondrial ROS, pro-inflammatory p-NF-κB and M1-genes, and suppressed anti-inflammatory p-AMPK, efferocytosis and M2-genes. Thus, our findings establish DRP1 sulfenylation as a previously unrecognized redox-sensing mechanism that links ischemia-induced ROS to reparative macrophage reprogramming and revascularization, identifying a novel therapeutic target for PAD. Full article

Background: Pancreatic ductal adenocarcinoma (PDAC) belongs to the group of killer human cancers. Its ferocity is sustained by an unusual mix of genetic changes—primarily in KRAS and TP53—a hypoxic as well as desmoplastic tumor microenvironment, plus metabolic and redox adaptations that allow tumor life amidst intense stress situations. Content: This paper will discuss the molecular networks of wild-type and mutant p53, wild-type and mutant KRAS, PUMA, TIGAR, PRMT5, NRF2, oxygen tension, reactive oxygen species (ROS), and oxidative stress pathways that contribute to pancreatic cancer. It will describe how these factors help set the tumor’s redox state and control apoptosis and therapeutic resistance. This shall therefore specifically discuss what role oxygen gradients play in pancreatic tissues, as well as retinoic acid, together with redox-targeted therapies that are specific to vulnerabilities within such types of networks. Summary and Outlook: An understanding of the crosstalk of these molecular pathways will be critical in designing rational therapeutic strategies. Genetics, metabolism, and microenvironmental integration may open a path toward combinatorial therapies that would resensitize PDAC to apoptosis and overcome resistance to current treatments. Full article

TP53 is the most frequently altered tumor-suppressor gene in human cancer, yet efforts to therapeutically target p53 have yielded limited and inconsistent clinical success. We argue that this gap reflects not a lack of druggable biology, but an oversimplified conceptual framework that treats p53 as a binary wild-type versus mutant entity. Here, we synthesize emerging evidence supporting a model in which p53 operates across a spectrum of functional states defined by mutation class, allelic burden, isoform composition, aggregation propensity, post-translational regulation, and cellular context. These states shape distinct biological outputs, including transcriptional activity, dominant-negative and gain-of-function effects, immune modulation, and checkpoint dependency, which collectively determine therapeutic vulnerability. We review current strategies targeting the p53 pathway, including mutant p53 reactivation, targeted degradation, anti-aggregation approaches, immune-directed therapies, restoration of wild-type pathway activity, gene replacement, and synthetic lethal targeting of DNA damage response dependencies. Clinical and preclinical evidence highlights key limitations of each approach, including stoichiometric constraints, mutation specificity, context-dependent efficacy, and adaptive resistance. Notably, emerging evidence from preclinical and correlative clinical studies suggests that therapeutic outcomes may be more closely associated with p53 functional state than with TP53 mutation status alone. We further emphasize the emerging roles of p53 isoforms and the tumor immune microenvironment as critical modifiers of p53 activity and determinants of treatment response. Collectively, these insights support a paradigm shift toward mechanism-matched, biomarker-stratified strategies that align therapeutic modality with the operative p53 network. Future progress will depend on integrating multi-parameter diagnostics with rational combination therapies to fully exploit p53 as a central vulnerability in cancer. Full article

APR-246 (Eprenetapopt) is a small-molecule drug that restores the activity of dysfunctional p53 proteins caused by missense mutations that affect the DNA-binding domain. However, recent studies suggest that APR-246 can also induce cell death in cancer cells that carry wild-type (WT) TP53. Here, we aimed to determine the impact of APR-246 on the survival of acute myeloid leukemia (AML) cells using isogenic Molm13 cells that harbor WT TP53, a missense mutation of TP53^R175H, or a biallelic deletion of TP53 (TP53^−/−). Our results showed that Molm13 TP53^−/− cells were significantly more resistant to APR-246-induced cell death compared with their Molm13 TP53^R175H/− mutant and Molm13 TP53^+/+ counterparts. In addition, knockdown of TP53 significantly reduced cytotoxicity induced by APR-246 in two TP53 WT AML cell lines (MV4-11 and OCI-AML2). Moreover, APR-246 markedly decreased the clonogenicity of TP53 WT hematopoietic stem/progenitor cells (HSPCs) isolated from humans and mice. In contrast, biallelic loss of TP53, but not TP53 missense mutation, significantly increased the resistance of mouse HSPCs to APR-246. Mechanistically, the loss of functional p53 proteins in Molm13 and MV4-11 cells decreased intrinsic apoptosis and impaired the production of cellular reactive oxygen species (ROS) induced by APR-246. Together, our results indicate that, in at least a subset of AML cell lines and normal HSPCs, APR-246-induced ROS production and cytotoxicity are enhanced in the presence of WT p53 proteins. Full article

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited targeted therapeutic options, underscoring the urgent need for novel treatment strategies. Feilike (FLK), a Traditional Chinese Medicine formula with heat-clearing and detoxifying properties, aligns with key pathological features implicated in breast cancer progression. In addition, several of its components have demonstrated anti-tumor activity, positioning FLK as a potential therapeutic candidate for TNBC. In this study, we employed an integrated approach combining network pharmacology, transcriptomic analysis, and experimental validation to investigate the anti-TNBC effects of FLK. Our results demonstrate that FLK significantly inhibits the proliferation of TNBC cell lines and patient-derived organoids and induces typical pyroptotic features, including cell swelling and increased lactate dehydrogenase (LDH) release. Mechanistically, FLK triggers a mutant p53 signaling cascade involving calcium dysregulation, endoplasmic reticulum stress (ERS) activation, mitochondrial dysfunction, and reactive oxygen species (ROS) accumulation, which collectively activate the P38/JNK–Caspase-3/GSDME pathway to induce pyroptosis. In vivo, FLK markedly suppresses tumor growth in a 4T1 orthotopic mouse model and enhances the anti-tumor efficacy of Cyclophosphamide. Furthermore, Licochalcone B (LCB) is identified as a key bioactive constituent that recapitulates the pyroptosis-inducing effects of FLK. Collectively, our findings uncover a previously unrecognized mutant p53–ERS–ROS–MAPK signaling axis underlying FLK-induced pyroptosis and provide mechanistic insight and experimental evidence supporting the repurposing of FLK as a potential therapeutic strategy for TNBC. Full article

Cancer is the second leading cause of death worldwide. Mutations in the TP53 gene lead to a loss of tumor suppressor function and an oncogenic gain of function for the protein, resulting in a more invasive, metastatic, and chemoresistant phenotype. Diverse structural studies have demonstrated that mutant p53 core domain unfolding is not irreversible. Thus, reactivation toward its wild-type-like conformation or inactivation of its mutant p53 capacities may restore the expression of genes in its tumor suppressor pathways, resulting in enhanced responses to current therapies. Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is a phytoalexin naturally found in more than 70 plant species that has widely proven antiproliferative and pro-apoptotic properties, as well as a capacity to reverse multidrug resistance in various cancer types. Interestingly, it has recently been demonstrated that resveratrol directly interacts with the p53 core domain and reduces mutant p53 aberrant aggregation. In this context, our study aims to elucidate whether resveratrol may induce antiproliferative and pro-apoptotic pathways regardless of a mutant background. We observed that resveratrol has an antiproliferative effect in cancer cells, independent of p53 status, and leads to apoptosis after 48 h of treatment. Resveratrol also induces the expression of p53 tumor suppressor target genes, which are involved in cell cycle arrest and apoptosis. Even though the previous effects are more significant in cells expressing wild-type p53, resveratrol drastically sensitizes all cancer cell lines, regardless of p53 status, to cisplatin treatment, making it a promising enhancer compound to overcome chemoresistance associated with p53. Full article

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and accounts for over 800,000 deaths worldwide, making it a major global health concern. Unfortunately, despite major advances in systemic treatments, such as the introduction of atezolizumab and bevacizumab, patient objective response rates fall below 30%. HCC most commonly develops against a background of chronic liver disease and cirrhosis, although single gene mutations can also drive HCC development, progression, and metastasis. Around 25% of HCC patient tumours carry mutations in TP53, the gene encoding the tumour-suppressor protein p53. p53 is a central regulator of genomic stability, cell-cycle arrest, apoptosis, senescence, and metabolic homeostasis, and its dysfunction is a frequent event in hepatocarcinogenesis. Accumulating evidence highlights the critical role of p53 in liver fibrosis, inflammation, and shaping of the HCC tumour microenvironment (TME). This review summarizes the role of p53 and its negative regulators Mdm2 and MdmX in HCC development and progression, with an emphasis on how p53 shapes the TME in favour of tumour progression. We also evaluate current and emerging p53-targeted therapeutic strategies, including Mdm2/MdmX inhibitors, mutant p53 reactivators, and rational combinations with immunotherapies. Finally, we discuss major challenges in translating p53-based therapies to the clinic, such as tumour heterogeneity, underlying liver dysfunction, and the development of therapeutic resistance. A deeper understanding of p53 biology in chronic liver disease may unlock new avenues for effective HCC prevention and treatment. Full article

Filamentous fungi exhibit high heavy metal resistance; elucidating their resistance mechanisms is of practical importance for fungal utilization and for engineering other microorganisms. However, the molecular basis of copper tolerance in filamentous fungi remains poorly understood, with few studies addressing this specific trait. Previously, we isolated a copper-hyper-resistant strain, P. janthinellum GXCR, and generated two mutagenized derivatives, EC-6 and UC-8. To investigate copper resistance, wild-type GXCR (WT) and mutants EC-6 and UC-8 were subjected to integrated physiological, biochemical, and transcriptomic analyses. Copper tolerance followed the rank order: WT > UC-8 > EC-6. Supplementation with Mn²⁺ or exogenous proline enhanced copper resistance. Under copper stress, intracellular reactive oxygen species (ROS) levels increased in all strains, correlating dynamically with activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as malondialdehyde (MDA) content, with all exhibiting a biphasic response: an initial rise followed by a decline with increasing Cu²⁺ concentration. WT accumulated less Cu and Cd but more Cr (at high concentration) than the mutants. In contrast, intracellular Pb accumulation in all three strains decreased monotonically with rising Pb doses. RNA-seq of WT and EC-6 grown in TYB with 0, 0.5 and 3 mM Cu²⁺ identified 8 copper-resistance-related genes, verified by real-time quantitative reverse transcription PCR (RT-qPCR). Weighted gene co-expression network analysis (WGCNA) clustered genes into 10 modules; integrating physiological data identified 10 traits, and the four most correlated modules yielded 116 hub genes mostly linked to energy metabolism, cell components and transporters. copA and ATP7, encoding Cu²⁺-exporting ATPases, were identified as central regulators of copper homeostasis and key contributors to enhance copper tolerance. These findings provide molecular insights into copper resistance of filamentous fungi and valuable genetic targets for rational strain engineering. Full article

Oxidative stress (OS) is generated by the imbalance between reactive oxygen species (ROS) and antioxidant enzyme activities, such as catalases, superoxide dismutases, and glutathione peroxidases. In the Y. lipolytica genome, three genes encoding catalases (CAT1, CAT2, and CAT3) have been identified; all three genes are transcriptionally active in cells grown under OS conditions. This study aimed to analyze whether the CAT1 and CAT2 genes exhibit a compensatory function that allows maintaining the functionality of the antioxidant response in Y. lipolytica cells lacking the CAT3 gene. The construction of the mutant strain (Ylcat3-Δ) was performed using Double-Joint PCR. OS was induced by the addition of H₂O₂ [5 mM], ROS production was quantified by fluorescence using 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and gene expression was analyzed by semi-quantitative RT-PCR in both parental (P01a) and mutant (Ylcat3-Δ) strains exposed or not to oxidative conditions. ROS production was lower in P01a cells than in Ylcat3-Δ cells when exposed to H₂O₂ [5 mM]. Also, under OS conditions, CAT1 gene expression levels decreased in both strains, whereas CAT2 gene expression increased in both types of cells. Under OS, both parental and Ylcat3-Δ strains showed similar growth rate, sensitivity to oxidative conditions and gene expression patterns, and it can be concluded that CAT3 gene deletion does not alter the transcriptional activity of CAT1 and CAT2 genes, suggesting that the compensatory function among the CAT genes of Y. lipolytica may not be limited to the presence/absence of CAT3 gene. Full article

Plants utilize cell surface pattern recognition receptors to recognize pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity (PTI) responses. Late blight, caused by the oomycete plant pathogen Phytophthora infestans, poses a major threat to global potato production. The oomycete PAMP, P. infestans cell wall ceramide D, triggers reactive oxygen species (ROS) production in potato and Arabidopsis. It is specifically recognized by the lectin receptor-like kinase RESISTANT TO DFPM-INHIBITION OF ABSCISIC ACID SIGNALING 2 (RDA2) in Arabidopsis. Treatment with P. infestans ceramide D enhances potato resistance against P. infestans. However, the function of RDA2 homologs in potato remains uncharacterized. Herein, potato RDA2 genes were identified through sequence alignment analysis. Their expression levels were subsequently measured in a potato inbred line infected with P. infestans. Notably, transient expression of StRDA2A, but not its kinase-dead mutant StRDA2A^K543M, caused cell death and enhanced disease resistance in Nicotiana benthamiana. Additionally, two RXLR-type effector proteins significantly inhibited StRDA2A-induced cell death. The findings of this study suggest that potato receptor kinase RDA2 proteins confer disease resistance, which is attenuated by RXLR effectors secreted by P. infestans. Full article

Osteosarcoma, the most prevalent primary malignant bone tumor in children and adolescents, is characterized by high rates of metastasis, recurrence, and chemotherapy resistance, leading to suboptimal patient survival. The MDM2-p53 pathway plays a pivotal role in its tumorigenesis and progression, where dysregulation leads to loss of p53 function. This review systematically elucidates the molecular mechanisms of this pathway and summarizes diverse targeted therapeutic strategies, including small-molecule MDM2 inhibitors, mutant p53 reactivators, and innovative modalities such as gene therapy and Proteolysis Targeting Chimeras (PROTACs). Despite demonstrating potent preclinical activity with low IC₅₀ values, the clinical translation of these agents has faced significant challenges. Early-generation MDM2 inhibitors (e.g., RG7112, Idasanutlin) showed limited monotherapy efficacy and dose-limiting toxicities like thrombocytopenia, halting their development at early-phase clinical trials. In contrast, novel MDM2 inhibitors like APG-115 have advanced to Phase II trials, marking a significant breakthrough. Although not yet tested in dedicated osteosarcoma cohorts, their safety and efficacy in MDM2-amplified solid tumors provide a critical foundation for the development of precision medicine and combination regimens for osteosarcoma. Future efforts to accelerate drug development may leverage single-cell sequencing and AI-aided drug design to decipher osteosarcoma heterogeneity and optimize drug profiles for reduced toxicity. Full article

Background: The turnaround of the tumor suppressor p53 protein, the guardian of the genome, is closely regulated to ensure avoidance of its untimely activation, which could lead to the demise of normal cells. Cancer cells often display mutations in the gene TP53 encoding for p53, which interferes with its normal function. Methods: The genomic series of colorectal cancer from the Cancer Genome Atlas (TCGA) was interrogated to discover genomic alterations and determine the mRNA expression of enzymes affecting p53 ubiquitination in colorectal cancers with wild-type and mutant TP53. Results: Genomic alterations of p53-regulating E3 ubiquitin ligases were uncommon in colorectal cancers, the most frequent being mutations in RCHY1. Several p53-regulating E3 ligases were well expressed in subsets of colorectal cancers, two of which, MDM2 and TRIM24, displayed higher mRNA expressions than the normal colorectal epithelia. The former was particularly upregulated in TP53 wild-type colorectal cancers, and the latter was upregulated in both wild-type and mutant TP53 cancers. Upregulation of TRIM24 in TP53 mutant cancers was observed independently of the type of mutations (gain-of-function or other). Among E3 ligases used in proteolysis-targeting chimeras (PROTACs), VHL was upregulated together with its E2-conjugating enzyme UBE2S in colorectal cancers. Conclusions: This survey of p53-targeting ubiquitin ligases provides a roadmap for potential therapeutic strategies working by promoting the destruction of the mutant protein or reactivating its normal function in TP53-mutated colorectal cancers and promoting p53 function by preventing degradation in TP53 wild-type cancers. Full article

Small heat shock proteins (HSP20s) are known to function as molecular chaperones that bind to denatured proteins under high-temperature stress and assist in their conformational recovery, thereby contributing to plant thermotolerance. In the present study, three HSP20 genes—PcHSP12.8, PcHSP12.9, and PcHSP13.4—were identified in the transcriptome of Polygonatum cyrtonema Hua. Bioinformatics analysis indicated their phylogenetic relationships, conserved domains, and potential tertiary structures. RT-qPCR analysis revealed up-regulation of all three genes in response to heat stress. Subcellular localization studies further suggested that PcHSP12.8, PcHSP12.9, and PcHSP13.4 are predominantly localized in the nucleus. Heterologous expression of these genes in a heat-sensitive yeast mutant appeared to improve cell survival under heat stress relative to the control strain. In Arabidopsis thaliana overexpressing these genes, moderate improvements in germination rate, root elongation, and stress survival were observed compared to wild-type plants under heat stress. Transgenic lines also showed a tendency toward reduced reactive oxygen species accumulation, as reflected by decreased 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, together with increased activities of catalase (CAT) and peroxidase (POD), as well as higher chlorophyll retention under thermal stress. Taken together, these findings imply that the three PcHSP20 genes could be involved in thermotolerance in P. cyrtonema. Full article

Human infections caused by pathogenic bacteria remain a major global health concern. Among them, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Salmonella typhi are particularly prevalent and associated with significant morbidity and mortality. While antibiotics have long been the cornerstone of bacterial infection treatment, the widespread and often inappropriate use of these drugs has led to the emergence of multidrug-resistant (MDR) strains. This escalating resistance crisis underscores the urgent need for alternative therapeutic strategies. Amid the escalating global antimicrobial-resistance crisis, a genome-wide screen of 1800 Saccharomyces cerevisiae knockouts identified a TAD1-deficient mutant whose cell-free supernatant (CFS) rapidly eradicates multidrug-resistant E. coli, S. aureus, K. pneumoniae, and S. typhi in vitro. CFS disrupts pathogenic biofilms, downregulates biofilm-associated genes, and exerts bactericidal activity by triggering intracellular reactive oxygen species (ROS) accumulation and compromising envelope integrity. Probiotic profiling revealed robust tolerance to an acidic pH and physiological bile, high auto-aggregation, and efficient co-aggregation with target pathogens. In both Galleria mellonella and murine infectious models, administration of CFS or live yeast significantly increased survival, attenuated intestinal histopathology, and reduced inflammatory infiltration. These data establish the TAD1-knockout strain and its secreted metabolites as dual-function antimicrobial-probiotic entities, offering a sustainable therapeutic alternative to conventional antibiotics against multidrug-resistant bacterial infections. Full article