Search Results (1,435)

The tumor suppressor protein p53, encoded by the TP53 gene, is known as the “Guardian of the Genome”, and alterations in TP53 are common to more than 50% of human cancers. p53 is a critical regulator of cellular responses to several stress conditions, such as DNA damage, oncogene activation, and nutrient starvation. p53 was traditionally described as a single transcription factor; however, now it is recognized as a complex family of isoforms generated through alternative promoter usage, alternative splicing, and alternative initiation of translation. These processes give rise to at least 12 distinct p53 isoforms in humans, including p53α (the canonical full-length isoform), p53β, p53γ, Δ40p53, Δ133p53, and Δ160p53, each with unique structural and functional properties. p53 isoforms differ in the presence or absence of specific and fundamental domains located both at N- and C-terminal ends, determining an altered DNA-binding potential, transcriptional activity, and protein–protein interactions. For instance, Δ133p53 isoforms lack part of the N-terminal domains and can exert dominant-negative effects over full-length p53α or modulate alternative transcriptional programs. Similarly, p53β and p53γ isoforms, which have a unique C-termini, influence cellular senescence. The expression patterns of p53 isoforms are tissue-specific and dynamically regulated under both physiological as well as pathological conditions. Alterations of isoform balance have been involved in tumor progression, metastasis, and therapy resistance. Importantly, specific isoforms can either enhance or limit canonical p53 tumor suppressor functions, thereby contributing to the functional diversity of the p53 network. Overall, the p53 isoform landscape adds a critical layer of complexity to p53 biology. In this review, we summarize the mechanisms underlying the production of p53 isoforms, their functions, and their expression in cancer, with the idea that a better understanding of the differential regulation and functional interplay of p53 isoforms may provide novel biomarkers and therapeutic targets in cancer. Full article

Glycosaminoglycans (GAGs) and their degrading enzymes have extensive applications and biotechnology and medicine, and play a crucial role in the recycling of organic matter in oceans. In this study, a potential GAG utilization gene cluster was identified in the genome of a novel marine Bacteroidetes, Roseihalotalea indica gen. nov. sp. nov. TK19036^T, through sole carbon source cultivation and differential proteomic analysis. Multiple GAG-lyases within this locus were purified and characterized. RiPL8 comprises a functionally unknown N-terminal domain and a catalytic C-terminal domain, exhibiting specificity for degrading hyaluronic acid (HA). The activity of RiPL35 is sensitive to Ca²⁺ ion concentration with an optimum at 10 mM. RiPL38 is the first reported member of the PL38 family capable of degrading HA and chondroitin sulfate (CS). In summary, our study reveals Roseihalotalea indica gen. nov. sp. nov. TK19036^T harbors an unpredicted GAG degradation gene cluster, and the encoded GAG-lyases exhibit distinct substrate specificities compared to the host organism. Full article

Melanoma is a highly malignant neoplasm of the skin with early metastatic spread and increasing incidence worldwide. Although there are significant therapeutic advances in immunotherapy, especially with the checkpoint inhibitors targeting PD-1 and CTLA-4, challenges such as treatment-related toxicities, a heterogeneous response to therapy, and drug resistance continue to exist. There are unmet needs for novel therapeutic strategies and/or approaches to complement the existing treatment options. Potential targets for future melanoma treatment are the gap junction proteins, connexins, which show an altered pattern of regulation during melanoma progression. In this review, we highlight the regulation of gap junctions during melanoma progression and the characterization of gap junctions as tumor suppressors during early-stage tumor development and then the reversion to enhancers of tumor metastasis during late-stage melanoma progression. We provide a comprehensive overview of gap junctions in the skin and how the connexin proteins, which comprise gap junctions, are alternatively regulated in melanoma progression. Connexins are protein channels in the human body that consist of 21 isoforms. These isoforms form gap junctions that provide important intercellular signaling and permeability channels. Each connexin protein consists of four transmembrane domains and a C-terminal tail, which is an important part of its function and regulation. Permeants of gap junctions include signaling molecules such as cyclic AMP and inositol triphosphate which are linked to key cellular behaviors such as proliferation and migration, making them essential for several tumor-related processes. At least ten connexin isoforms are found in normal skin. Connexin 43 (Cx43) is classified as the most prevalent isoform while Connexin 26 (Cx26) has been reported to be more specialized with restricted expression patterns. Cx43 and Cx26 regulate the growth, differentiation, and repair of the epidermis after injury. Evidence suggests that connexins have a stage-related function in melanoma. Loss of connexin expression and gap junctional intercellular communication is linked to tumor suppression and loss of differentiation in early-stage melanoma, while re-expression or overexpression of specific connexins, notably Cx43, may promote metastasis through enhanced tumor–stromal interactions and increased motility in late-stage melanoma. Such opposing actions of connexins support their candidacy as biomarkers and therapeutic targets. Understanding the dual-stage related functions of connexins in melanoma development and progression may lead to less cytotoxic and more efficient future therapeutic approaches. Full article

Background/Objectives: Bovine ephemeral fever (BEF) is a significant disease affecting the cattle industry. The current control strategy for BEF in the field primarily relies on inactivated vaccines. However, some individuals have experienced hypersensitive reactions to these vaccines, prompting the exploration of subunit vaccines as a potential alternative for BEF prevention. Glycoprotein (G protein)-based subunit vaccines derived from virions have successfully induced neutralizing antibodies in cattle for over a decade. Nevertheless, the lack of recent studies evaluating their efficacy using recombinant proteins has raised concerns regarding the development of BEF subunit vaccines for practical field application. Therefore, the objective of this study was to evaluate the antigenicity of a novel truncated G protein produced in mammalian cells as a candidate subunit vaccine for BEF in cattle. Methods: In this study, the G protein with full ectodomain and a version truncated at the C-terminal domain were successfully generated using the ExpiCHO™ expression system. Vaccine efficacy was evaluated weekly by measuring neutralizing antibody titers and cytokine mRNA expression levels following vaccination. Results: Results show that the recombinant protein s510, derived from the G protein of BEF, can stimulate cattle to produce an average 35-fold increase in neutralizing antibodies after three doses of vaccination. The significant upregulation of IFN-γ mRNA supports the effectiveness of the s510-based subunit vaccine and indicates the activation of a cytotoxic immune response in cattle following vaccination. Conclusions: In conclusion, the results indicate that the recombinant protein s510 is a promising antigen for future BEF subunit vaccine development in this study. Full article

DaxibotulinumtoxinA for injection (DAXI) is a botulinum neurotoxin (BoNT) drug product comprising the 150 kDa pure BoNT/A1 as the drug substance formulated with a proprietary stabilizing excipient, RTP004. We hypothesized that RTP004 facilitates localization of BoNT/A1 to the neuronal membrane, resulting in increased BoNT internalization and cleavage of the synaptosomal-associated protein of 25 kDa (SNAP-25) within synaptic terminals. We characterized the interaction between RTP004 and BoNT/A1 using in silico and in vitro techniques. In vitro analyses revealed that negative charges on the BoNT/A1 surface were located on the light chain (LC, the catalytic domain) and the C-terminus of the heavy chain (H_C, the receptor-binding domain), potentially providing sites for interaction with the positively charged RTP004 peptide. RTP004 bound to BoNT/A1, but not to human serum albumin (HSA), in both static and dynamic conditions. RTP004, not HSA, enhanced binding of BoNT to artificial membranes and RTP004 dissociated from BoNT under conditions that mimicked physiological conditions of the synaptic vesicle. RTP004 also increased binding of BoNT to the synaptosomal cell membrane and enhanced cleavage of SNAP-25 in a dose-dependent manner. These findings demonstrate that RTP004, not the excipient HSA common in other BoNT/A1 drug products, enhances binding of BoNT to the cell surface, facilitates internalization of BoNT into the cell, and increases SNAP-25 cleavage. Full article

Apolipoprotein E4 (apoE4) is the strongest genetic risk factor for late-onset Alzheimer’s disease (AD). Yet the molecular mechanisms underlying its contribution to AD remain to be fully elucidated. Here, we developed and characterized a set of apoE-specific single-domain antibodies (nanobodies) as a molecular toolbox to investigate intracellular apoE4. The nanobodies bind human apoE with nanomolar to sub-nanomolar affinity and recognize both apoE3 and apoE4. Domain-level epitope mapping revealed nanobodies that selectively bind either an N-terminal (residues 1–173) or C-terminal (residues 170–299) apoE4 fragment. Several nanobodies were validated as endoplasmic reticulum-targeted intrabodies that bind apoE4 intracellularly and promote its intracellular retention. These nanobodies constitute a versatile toolbox for probing and manipulating apoE4 in cellular models. As an exploratory application of this nanobody toolbox, we examined cytosolic apoE4, motivated by previous studies suggesting that cytosolic apoE4 fragments may influence AD-related cellular processes. Cytosolic expression of apoE4 resulted in perinuclear protein assemblies and the appearance of a ~25 kDa apoE4 fragment. Using a nanobody-based nuclear relocalization assay, we showed that cytosolic apoE4 remains accessible for nanobody binding but was not relocated to the nucleus by a nuclear localization signal-equipped nanobody. Altogether, this study introduces a nanobody-based toolbox to investigate apoE4 in distinct intracellular contexts, which can be relevant to AD. Full article

Rice (Oryza sativa) is a staple crop that is highly susceptible to heat stress (HS), which severely impairs growth and yield. In this study, we identified the rice Ovate Family Protein OsOFP3 as a novel negative regulator in response to heat. Our results demonstrate that the expression of OsOFP3 is suppressed at both the transcriptional and protein levels under HS. Overexpression of OsOFP3 significantly reduces the survival rate of rice seedlings under HS and exacerbates chlorophyll degradation, membrane damage, and the accumulation of reactive oxygen species (H₂O₂ and O₂⁻). In contrast, OsOFP3 mutants exhibit enhanced heat tolerance. Moreover, OsOFP3-overexpressing plants display increased stomatal opening and decreased stomatal closure under HS. Molecular interaction analysis further reveals that OsOFP3 interacts with the C-terminal domain of OsHTAS, a known positive regulator of heat tolerance encoding an E3 ubiquitin ligase, and this interaction depends on the RING domain of OsHTAS. Taken together, our findings indicate that OsOFP3 negatively regulates rice heat tolerance by disrupting ROS homeostasis, inhibiting stomatal closure, and potentially antagonizing the OsHTAS-mediated signaling pathway. This research provides new insights into the molecular mechanisms underlying HS tolerance in rice. Full article

Rapid antigenic drift in the coronavirus spike protein motivates alternative antiviral strategies. We target the conserved nucleocapsid (N) protein—central to RNA binding, genome packaging, and replication—and perform a comparative, cross-species 3D structure-based in silico evaluation. A library of 494 compounds (natural, phytochemical, synthetic) was docked with AutoDock Vina against the MERS-CoV N–terminal RNA–binding domain (NTD; PDB 7DYD) and the C–terminal dimerization domains (CTD) of SARS-CoV (2CJR) and SARS-CoV-2 (8R6E), reflecting the availability of high-resolution, functionally relevant domain structures for each virus. Top-ranked poses underwent ADME profiling and 100 ns GROMACS molecular-dynamics (MD) simulations. Myricetin 3-O-β-D-Galactopyranoside (myricetin) showed the most favorable predicted docking scores across targets (−8.9 kcal/mol, MERS–NTD; −10.1, SARS–CTD; −9.8, SARS-CoV-2 CTD). Curcumin showed moderate predicted affinity (−7.1 to −8.1), while MCC950 achieved consistently favorable docking score (−7.9 to −9.0). ADME results highlighted a trade-off: glycosylated flavonoids offered rich interaction networks but violated oral drug-likeness criteria (e.g., high TPSA), whereas MCC950 met Lipinski/Veber guidelines, supporting translational potential. MD analyses revealed ligand- and target-specific stability: myricetin maintained persistent binding over 100 ns in the SARS-CoV-2 CTD with lower RMSD than comparators; curcumin exhibited transient stability (~30 ns) in MERS- and SARS-bound complexes; MCC950 showed intermittent interactions. Collectively, these findings suggest that the conserved N protein RNA-binding groove represents a resistance-resilient target for broad-spectrum antiviral discovery. Natural flavonoids provide promising scaffolds for optimization, and MCC950 warrants further exploration given its drug-like profile. As this study is purely computational, the results are hypothesis-generating and should be validated via RNA-binding disruption assays, antiviral cell studies, and in vivo models. Full article

Cyclin-dependent kinase 1 (CDK1) regulates multiple cellular processes that HSV-1 can exploit to promote its own replication, particularly during the early steps of lytic infection. We investigated whether CDK1 inhibition disrupts immediate-early (IE) gene expression and analyzed the host phosphoproteome early in infection to identify putative host factors and mechanisms that facilitate HSV-1 IE gene expression and are controlled by CDK1. Human foreskin fibroblasts (HFFs) were pre-treated with a CDK1 inhibitor and showed a 1000-fold reduction in HSV-1 replication and significant reductions in IE mRNAs and protein levels at 4 hpi. We characterized cells after CDK1 inhibition and HSV-1 infection at 3 hpi by tandem mass spectrometry and identified >5500 phosphopetides (~2600 proteins), analyzing differential phosphorylation and protein–protein interactions. We validated CDK1 inhibition by detecting phosphorylation-specific decreases in known CDK1 substrates, as well as Robust Kinase Activity Inference. Rank- and network-based analyses of our dataset highlighted several candidate proteins, linking their CDK-directed phosphorylation to HSV-1 IE gene expression. Notably, the C-terminal domain of the large subunit of RNA polymerase II (RNAPII), POLR2A, is extensively phosphorylated, and its phosphorylation is significantly reduced upon CDK1 inhibition during viral infection. Taken together, these data support a model in which CDK1 activity maintains a transcriptionally permissive cellular state required for efficient HSV-1 IE gene expression. Our data suggest that when CDK1 is pharmacologically inhibited, key transcriptional facilitators are dysregulated, impairing viral transcription and replication. Full article

The TonB-dependent receptors (TBDRs) FepA and FhuA transport the siderophores ferric enterobactin (FeEnt) and ferrichrome (Fc), respectively, through the Gram-negative bacterial outer membrane. Their uptake mechanism involves conformational change in an ~150 residue N-terminal luminal domain (NTLD), located within their C-terminal β-barrel (CTβB) channels. We identified four internal sites (1–4) in TBDR that form a conserved network of ion pairs encircling the NTLD-CTβB interface. We tested the mechanistic importance of these electrostatic interactions by engineering systematic Ala substitutions in FepA and FhuA for the acidic or basic side chains that comprise them. Siderophore nutrition assays, colicin susceptibility tests and fluorescence spectroscopic uptake measurements of the mutants showed the importance of site-2, that adheres the base of NL1/Nβ3 and Nβ5 of the NTLD to β14 and β17 on the interior of the CTβB. Disruption of electrostatic bonds at site-2 reduced or eliminated ferric siderophore uptake and severely curtailed colicin susceptibility. Despite these reductions in ligand transport, fluorescent spectroscopic binding measurements showed that the site-2 mutations did not alter the affinity of FepA for FeEnt, nor FhuA for Fc. Elimination of ionic interactions at the three other locations in FepA (sites-1, -3, -4) did not reduce FeEnt uptake. Lastly, the disruption of ionic bonding at site-2 in FepA rendered it more susceptible to proteolysis, in part by OmpT, suggesting that ablation of ionic interactions in site-2 destabilized the NTLD within the CTβB. Overall, the experiments demonstrated that the ion pairs at site-2 in FepA and FhuA, that are evolutionarily conserved in the TBDR superfamily, are essential to the movement of ferric siderophores through the CTβB into the periplasm. Full article

Nanobodies (VHHs or single-domain antibodies) are powerful affinity reagents, but their routine use is often limited by production constraints and by the lack of a conserved Fc region for secondary detection. We describe a simplified strategy in which functional GST–nanobody fusion proteins are expressed directly in the cytoplasm of Escherichia coli Origami^TM 2 (DE3), a strain that supports disulfide bond formation through trxB/gor mutations. Using well-characterized nanobodies against GFP (Lag2) and mCherry (C11), we designed N-terminal GST fusions and confirmed by AlphaFold3-based modeling that both constructs preserve the GST fold and the VHH (Variable domain of the Heavy-chain antibody of Heavy-chain-only antibodies) β-sandwich with defined CDR loops and a predicted intradomain disulfide bond. Following IPTG induction and purification by glutathione affinity and size-exclusion chromatography, we obtained soluble GST-nb-GFP and GST-nb-mCherry at ~8–12 mg/L. Isothermal titration calorimetry showed nanomolar binding to their antigens (K_d ~123 nM for GFP and ~199 nM for mCherry). Consistent with conformational epitope recognition, GST-nanobodies were reactive in native-state dot blots but not in denaturing Western blots under the conditions tested. The GST moiety enabled indirect immunofluorescence via anti-GST antibodies, yielding specific labeling of GFP- or mCherry-tagged TGN38 in HeLa and H4 cells. Finally, we demonstrate “GST-nanobody pulldown” as a robust method for affinity capture from cell lysates. Together, this platform provides a low-cost, versatile route to functional nanobody reagents without requiring tag removal, and complements other nanobody designs (e.g., VHH-Fc fusions) in an application-dependent manner. Full article

To address coverage gaps in high-latitude space weather monitoring caused by constraints in energy, bandwidth, and labeled samples, this study presents a systematic solution deployed in Hailar, China. We constructed a Cloud–Edge–Terminal system featuring wind–solar hybrid energy and RK3588-based edge computing, achieving six months of stable ionospheric–geomagnetic observation under −40 °C. Furthermore, we propose a Dual-Adversarial Recurrent Autoencoder (DA-RAE) for anomaly detection. Utilizing a single-source domain strategy, the model learns physical manifolds from quiet-day data, enabling zero-shot anomaly perception in the unsupervised target domain. Field tests in March 2025 demonstrated superior generalized anomaly detection capabilities, successfully identifying both transient space weather events and environmental equipment faults (baseline drifts). This work validates the value of edge intelligence for autonomous operations in extreme environments, providing a reproducible paradigm for global ground-based networks. Full article

Hepatitis C virus (HCV) chronically infects over 50 million people worldwide and poses a significant risk to global health. The HCV NS3/4A complex, a bifunctional enzyme comprising a protease and a helicase domain, is indispensable for viral replication and immune evasion, making it a pivotal target for direct-acting antiviral agents (DAAs). Here, we summarize its structural features, functional mechanisms, and implications in drug design and protein engineering (e.g., nanopore sequencing applications). The NS3 protease domain is activated by the NS4A cofactor, which mediates viral polyprotein processing and relies on a zinc-binding site for structural stability. The C-terminal helicase domain catalyzes ATP-dependent 3′→5′ unwinding, and allosteric crosstalk between the protease and helicase domains dynamically modulates the enzymatic activity, balancing unwinding velocity and processivity. Beyond supporting viral replication, NS3/4A cleaves MAVS to abolish RIG-I/MDA5 signaling but spares TRIF, leaving TLR3-mediated immunity intact; it also modulates host lipid and iron metabolism, contributing to HCV pathogenesis. Notably, structural and functional studies of NS3/4A lay a solid theoretical foundation for developing novel therapeutic strategies. Currently, DAAs targeting NS3/4A have achieved high sustained virologic response rates; however, resistance-associated substitutions remain a major clinical challenge, particularly in genotype 3 infections. Emerging therapeutic strategies targeting NS3/4A include allosteric inhibition and proteolysis-targeting chimeras (PROTACs)-mediated degradation. Full article

The p53 tumor suppressor protein plays a central role in maintaining genomic stability by regulating cell cycle arrest, apoptosis, and DNA repair under cellular stress. Mouse double minute 2 (MDM2), an E3 ubiquitin ligase, negatively regulates p53 via direct binding and proteasomal degradation. Overexpression or amplification of MDM2 can disrupt this pathway and promote tumorigenesis, even in cancers with wild-type p53. This review outlines the structural features of MDM2, particularly its N-terminal hydrophobic pocket and C-terminal RING domain, and their roles in p53 regulation. We further examine the pathological effects of MDM2 dysregulation and SNPs linked to increased cancer risk. Recent progress in small molecule MDM2 inhibitors is discussed, with a focus on non-covalent agents such as rhein-derived anthraquinone analogs, including AQ-101, which demonstrate promising anti-cancer activity with reduced toxicity. These findings support the continued development of non-covalent MDM2 inhibitors as a novel therapeutic approach for cancers involving both wild-type and mutant p53. Full article

Phytochelatin synthases (PCSs) are pivotal enzymes in heavy metal detoxification, yet also implicated in sulfur homeostasis and redox regulation. In this study, we report the molecular and functional characterization of the PCS gene from the green alga Scenedesmus acutus (SaPCS), comparing wild-type and chromium-tolerant strains of this microalga. RT-qPCR, immunoblotting and mass spectrometry analyses revealed that SaPCS expression and protein abundance are primarily regulated by sulfur availability rather than by chromium stress. Two protein isoforms (~70 kDa full-length and ~34 kDa truncated) were detected, both more abundant in the chromium-tolerant strain than the wild-type and responsive to sulfur availability. Furthermore, three alternatively spliced transcript variants (SaPCSa, SaPCSb, SaPCSc) lacking the C-terminal domain coding region but retaining a functional or partially disrupted N-terminal catalytic domain were identified, contributing to the post-transcriptional diversification of PCSs. Mass spectrometry analyses showed negligible phytochelatin production in response to chromium treatment, indicating that detoxification of this metal in S. acutus relies mainly on glutathione (GSH) conjugation and the ascorbate–GSH antioxidant cycle. Overall, these results suggest that SaPCS may promote chromium tolerance by modulating sulfur and redox metabolism rather than by driving phytochelatin accumulation, highlighting the remarkable functional plasticity of PCSs in algal stress responses. Full article