Cathelicidin-DM is a host defence peptide isolated from the Asian common toad Duttaphrynus melanostictus. The peptide has been reported in the literature to exhibit antibacterial and wound-healing activities. The mechanism of antimicrobial activity, however, remains unclear. As most cathelicidins reported in the literature exhibit their antimicrobial activity through membrane perturbation, we investigated cathelicidin-DM's interfacial and membrane binding activities using molecular dynamics simulations and experimental methods that include surface activity at air/aqueous interface, binding with liposomes using fluorescence spectroscopy, binding with lipid monolayers using Langmuir set-up, and dye release assay. The molecular dynamics simulation of cathelicidin-DM in water/cyclohexane biphasic system reveals its localization at the water/cyclohexane interface. The experiment shows a saturation surface pressure of ∼10-11 mN/m at the air/aqueous interface, thereby establishing its interfacial activity. The simulation with the POPC/CHL bilayer results in extensive unfolding of the peptide, with the peptide showing transient interactions with the bilayer. With POPE/POPG bilayer, the peptide binds through its N-terminus and remains bound throughout the simulation. Lipid monolayer penetration assays with POPC/CHL (10:1) and POPE/POPG (7:3) monolayers exhibited critical insertion pressures of ∼33.2 and ∼46.7 mN/m, respectively. Preferential binding to negatively charged membranes is further established using tryptophan fluorescence assays. Counterintuitively, however, the peptide preferentially perturbs zwitterionic vesicles, sparing the negatively charged ones. Antimicrobial peptide molecules usually act cooperatively to bring about the membranolytic effects. Tight binding to membranes could, therefore, affect cooperativity, thereby negatively impacting activity.

Due to the invasive nature of cancer cells, therapies targeting a single cell death pathway alone often encounter drug resistance; therefore, the development of novel therapeutic strategies, such as the dual induction of apoptosis and ferroptosis, is warranted. This study investigated the anticancer mechanism of the newly synthesised thiolato-bridged manganese-based CO-releasing molecule (S-MnC) in highly metastatic pancreatic (PANC-1) and prostate (PC-3) cancer cells. The myoglobin assay showed controlled liberation of approximately 1.5 CO equivalents per S-MnC molecule in the dark, and approximately 1.1 equivalents under UV irradiation, consistent with a sustained single-donor profile. S-MnC induced apoptosis through ROS generation via Fenton-like reaction, mitochondrial membrane disruption, caspase activation, and DNA damage, culminating in G1-phase cell cycle arrest. Ferroptosis was triggered concurrently via p53-mediated suppression of SLC7A11 and GPX4, leading to GSH depletion, and iron overload was evident, with upregulated TFR1 expression. Notably, a crosstalk between these two cell death pathways was demonstrated, as ferrostatin pretreatment rescued the expression of apoptotic genes, suggesting that ferroptotic signalling actively contributes to the apoptotic response induced by S-MnC. Moreover, S-MnC also exhibited antimetastatic potential by suppressing MMP-2 and MMP-9. S-MnC showed promising binding potential towards pancreatic and prostate cancer-specific targets, mesothelin and prostate cancer-specific membrane antigen, with predicted binding energies of -8.4 and -9.1 kcal/mol, respectively. Our results, for the first time, establish that S-MnC is a potent, dual-action therapeutic agent that mediates both apoptosis and ferroptosis, offering a promising strategy to overcome drug resistance in aggressive, metastatic cancers and to enhance therapeutic efficacy.

β-Escin is a well-known saponin derived from the horse chestnut tree. Saponins, including escin, exhibit a wide range of biological activities, among which membrane-modifying activity is particularly notable. The latter is key to understanding the molecular mechanisms underlying escin's biological effects, including its antiviral properties. For example, altering membrane properties can strongly influence the life cycle of enveloped viruses such as SARS-CoV-2 by modulating the activity of its envelope protein (E protein). In the present paper, we studied the localization of both escin and the E protein transmembrane domain (ETM) in model membranes with different lipid compositions. We demonstrated that escin can alter ETM localization in membranes containing the negatively charged lipid DMPS, and that escin is also capable of extracting cholesterol from model lipid membranes.

tRNA-derived fragments (tRFs) have arisen as critical regulators in cancer development, however, their functions in esophageal squamous cell carcinoma (ESCC) remain poorly understood. Here, we reported that tRF-17-9L5FZU3 was significantly upregulated in ESCC cells and tissues, and correlated with poor patient outcome. Through integrated small RNA sequencing and qRT-PCR validation in 40 paired clinical samples, functional analyses, including colony formation, CCK-8, Transwell migration/invasion assays, and xenograft mouse models, demonstrated that tRF-17-9L5FZU3-knockdown suppresses ESCC cell proliferation, invasion, migration, and tumor growth in vivo. Furthermore, tRF-17-9L5FZU3 was shown to directly bind the 3'UTR of RhoB (Ras Homolog Family Member B) mRNA via dual-luciferase reporter assays, RNA pull-down, and AGO2 co-immunoprecipitation, leading to repression of RhoB expression at both mRNA and protein levels. RhoB was consistently downregulated in ESCC tumors and exhibited a significant inverse correlation with tRF-17-9L5FZU3 expression. Rescue experiments confirmed that oncogenic effects of tRF-17-9L5FZU3 were dependent on RhoB suppression. Moreover, tRF-17-9L5FZU3-knockdown decreased TGF-β secretion, increased IL-2 production, and inhibited PI3K/AKT and NF-κB signaling, which were reversed by co-silencing of RhoB. Collectively, these findings found that tRF-17-9L5FZU3 promoted ESCC malignancy by directly repressing RhoB, thereby activating oncogenic signaling pathways, positioning the tRF-17-9L5FZU3-RhoB axis as a critical regulator and potential therapeutic target in ESCC.

UDP-galactose 4-epimerase (Gal4E) catalyze the reversible 4-epimerization of UDP-d-galactose to UDP-d-glucose, a crucial step in galactose metabolism first described by Luis Leloir. Since its initial discovery, a plethora of homologous 4-epimerases has been reported with activities on a variety of other nucleotide sugars, including UDP-D-N-acetyl-galacto/glucosamine, UDP-xylose/l-arabinose, UDP-galacturonic/glucuronic acid, dTDP-l-rhamnose/6-deoxy-l-talose and/or GDP-hexoses. These NDP-sugar 4-epimerases (NS-4Es) play critical roles in diverse carbohydrate metabolic pathways across all domains of life and exhibit conserved structures and mechanism. Hence, various models have sought to predict specificity by focusing on steric constraints and conserved motifs, with successful specificity switches in Gal4Es based on and supporting the gatekeeper model. However, mutational analysis in other representatives also challenge these models, suggesting that additional factors like molecular interactions and conformational dynamics may significantly influence substrate specificity. This study combines sequence similarity networks, site saturation mutagenesis and molecular dynamics to investigate substrate specificity in NS-4Es. We propose an expanded classification, increasing specificity groups from three to seven to incorporate epimerases active on GDP-sugars (group 4), UDP-d-xylose (group 5), UDP-d-galacturonic acid (group 6), and dTDP-l-rhamnose (group 7). Mutagenesis of the gatekeeper in GDP-hexose 4-epimerase PhGal4E_1 revealed enhanced activity on GDP-D-Man, which was found to be driven both by gatekeeper size and polarity as well as by altered active site interactions. Molecular dynamics confirmed a dynamic network and clamping down mechanism within the active site as key determinants of specificity across NS-4Es, offering new insights into their functional diversity.

Protocatechuic acid (PCA) is both a prominent gut microbiota-derived metabolite of anthocyanins and a naturally occurring phenolic acid presents in various edible plants. Although this compound has been reported to exert cytotoxic effects at millimolar concentrations in vitro, its plasma levels following dietary intake are limited to the non-toxic nano-to micromolar range. Here, we identify the adaptor protein TNF Receptor Associated Factor 2 (TRAF2) as a direct molecular target of this phenolic compound. By computational modeling and biophysical assays, including fluorescence spectroscopy and calorimetry, we demonstrate that PCA directly binds the TRAF domain of TRAF2, leading to decreased thermal stability and potential alteration of trimeric interfaces. By performing functional analyses in HAP1 leukemia cells, we provide evidence that PCA, at low micromolar concentrations, increases cell viability through a TRAF2-dependent mechanism. Mechanistically, PCA binding to TRAF2 inhibits JNK signaling while redirecting TRAF2 toward pro-survival signaling routes involving c-Myc. Collectively, these findings uncover TRAF2 as a novel molecular target of PCA and provide a mechanistic basis for its effects on cell viability.

Human hepta-helical transmembrane spanning G protein-coupled family of dopamine receptors (DRs) play key neurophysiological roles and are involved in the pathogenesis of Parkinson's disease due to dopamine deficiency. Previous studies found that halogen doping can be used as a chemical strategy to control the optical and spectroscopic properties of dopamine molecule and to shift the binding mode of various exogenous compounds to DRs. In this study, we attempted to investigate the halogen-doping effects by substituting the three positions (X1, X2 and X3) of dopamine's aromatic phenyl group with four halogen atoms (F, Cl, Br and I) on the binding affinity and recognition specificity of halogen-doped dopamine analog ligands to five DR receptors. A systematic halogen-doping substitution profile was created computationally, from which a number of very favorable substitutions (VFSs) and very unfavorable substitutions (VUSs) were identified, and their conformational and energetic properties were examined in the modeled complex structures with DRs, revealing that some VFSs can be attributed to the formation of additional halogen bond (X-bond) or hydrogen bond (H-bond) between DR receptors and halogen-doped analog ligands upon the substitutions. Fluorescence analysis observed that the dopamine binds potently to D1 and D2, with a moderate selectivity (⌂KD[D2/D1] = 0.5-fold) for D2 over D1, which can be improved considerably to D1 upon the iodine substitution at X2 position (X2-I) or upon the bromine substitution at X3 position (X3-Br), but the two substitutions have only a moderate effect on dopamine affinity to D2. Consequently, the dopamine selectivity between D1 and D2 was reversed by the two substitutions, that is, the selectivity was changed from D2-over-D1 (native dopamine, ⌂KD[D2/D1] = 0.5-fold) to D1-over-D2 (⌂KD[D2/D1] = 18.9-fold upon X2-I substitution and 1.8-fold upon X3-Br substitution). This work demonstrated that the binding affinity and recognition specificity of dopamine ligand to different DR receptors can be rationally altered by careful design of halogen types and their substitution positions on dopamine's aromatic ring.

Apolipoprotein E (ApoE) is a key regulator of cholesterol transport in both the periphery and the central nervous system and is the major genetic risk factor for late-onset Alzheimer's disease. Although ApoE deficiency is known to elevate circulating cholesterol levels, its consequences for brain cholesterol homeostasis, synaptic integrity, and cortical network excitability remain incompletely understood. In the present study, ApoE knockout (ApoE[-/-]) and wild-type (WT) mice were used to evaluate systemic and brain cholesterol levels, synaptic protein expression, and cortical electrophysiological activity. ApoE deficiency resulted in significantly elevated serum cholesterol levels compared with wild-type mice. In contrast, cortical cholesterol levels were significantly reduced in ApoE[-/-] mice, whereas hippocampal cholesterol levels remained unchanged. Consistent with altered cortical lipid homeostasis, synaptophysin (SYP) and postsynaptic density protein-95 (PSD-95) levels were significantly decreased in ApoE-deficient animals. At the functional level, ApoE deficiency was associated with reduced basal cortical network activity, reflected by lower electrocorticographic (ECoG) power. Moreover, pharmacological excitation with 4-aminopyridine (4-AP) produced a markedly attenuated increase in cortical excitability in ApoE-deficient mice. Inflammatory markers, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), showed no differences between WT and ApoE[-/-] mice. Similarly, oxidative stress markers measured in cortex and hippocampus did not indicate a consistent increase in oxidative damage. These findings demonstrate that ApoE deficiency is associated with a multilevel disruption characterized by elevated peripheral cholesterol, reduced cortical cholesterol availability, synaptic protein loss, and diminished cortical network excitability under both basal and hyperexcitable conditions. The results highlight a potential link between impaired brain cholesterol homeostasis and altered synaptic network function in the cortex of ApoE-deficient mice.

This study analyzed an effect of nine different zinc (Zn) concentrations on the structure and function of pigment-protein complexes in the thylakoids of Phaseolus coccineus exposed to Zn concentrations ranging from 25 to 1000 μM. Zn exposure induced non-linear concentration-dependent changes in Lhcb1 and Lhcb2 phosphorylation, accompanied by alterations in 77 K fluorescence emission and fluorescence lifetime parameters, indicating reorganization of the LHCII antenna and changes in the excitation energy distribution within the photosynthetic apparatus. Fluorescence lifetime analyses showed that 100 μM Zn[2+] is associated with changes in the relative amplitudes of decay components, including a decrease in the 70 ps component and an increase in the 1.6 ns and 0.28 ns components, suggesting modifications in energy transfer dynamics and possible partial uncoupling between antenna complexes. However, decay-associated lifetimes are not uniquely diagnostic, and alternative interpretations cannot be excluded. The highest Zn[2+] concentration (1000 μM) was associated with pronounced alterations in pigment-protein organization in the thylakoid membranes, supported by Fourier-transform infrared analysis showing the disappearance of the band around 1590 cm[-1]. Changes in carotenoid and chlorophyll content appear to play an important role in the functioning of the photosynthetic apparatus of P. coccineus under heavy metal stress. Overall, Zn treatment induced coordinated, multi-level reorganization of the thylakoid membrane, involving changes in LHCII phosphorylation, oligomeric state, excitation energy distribution, and fluorescence lifetime characteristics. Together, these datasets reveal a dynamic and non-linear response of the photosynthetic apparatus to increasing Zn concentrations, reflecting both regulatory acclimation and stress-induced structural remodeling.