Biochimica et Biophysica Acta (BBA) - General Subjects

In TMPRSS2 fusion-positive prostate cancer, ERR is involved in regulation of ERG and promotes the androgen receptor independent signaling in the cancer progression. The ERR binds to the ERREs (estrogen-related receptor response elements) present at -5042 bp of the TMPRSS2- promoter and enhances the ERG overexpression that causes prostate cancer progression. To dissect the structural basis of the ERR recognition to the TMPRSS2 promoter DNA, we have purified the full-length ERR (ERRFL), NTD deleted construct (ERR{Delta}NTD), and the DNA-binding domain (ERRDBD) proteins and performed the binding analysis with 30 bp TMPRSS2-promoter DNA (5' -AGTCCAAGGTCGGTGGATC ACAAGGTCAGG-3'). Circular dichroism analysis showed that all three ERR proteins adopt native secondary structures. DNA binding induced subtle changes in the secondary structures, while enhancing the thermal stability (Tm) of all ERRa proteins. Binding analysis showed that ERRDBD bound weakly to the DNA, whereas ERRFL and ERR{Delta}NTD exhibited substantially higher affinities ~120-fold and ~131-fold than ERRaDBD, respectively. Small-angle X-ray scattering (SAXS) analyses revealed a dimeric ERRFL structure and an ERRFL-DNA complex (2:1) structure in solution and fitted well with Alpha Fold model of apo and DNA bound complex of ERRFL. Furthermore, 100 ns dynamics simulations on apo and DNA-bound ERRa proteins showed that all proteins remained structurally stable, with flexibility largely confined to loop regions of ERRa proteins. Our biophysical, DNA binding and structural analyses have revealed the mechanism involved in ERR recognition of the TMPRSS2- promoter DNA, which provides insight into ERR-mediated transcriptional regulation and development of anticancer drugs against ERR-driven prostate cancer.

Histone deacetylases (HDACs) are pivotal epigenetic regulators that modulate diverse cellular pathways by removing acetyl groups from lysine residues on both histone and non-histone proteins. Histone deacetylase 11 (HDAC11), the sole member of class IV HDACs, exhibits both deacetylation and fatty acid deacylation activities. Accumulating evidence implicates HDAC11 as a key epigenetic regulator of fundamental cellular processes, including metabolism, immune responses, and tissue development. Dysregulation of HDAC11 activity has been associated with inflammatory diseases, metabolic disorders, neurodegenerative conditions, and cancer, highlighting its potential as a therapeutic target. Although several HDAC11-specific inhibitors have been identified, none have progressed to clinical development. In this study, we aimed to discover HDAC11-selective inhibitors by integrating in silico and in vitro validation approaches. Homology modelling of the HDAC11 structure was conducted, followed by model validation, structure-based virtual screening, molecular dynamics (MD) simulations, and binding free energy calculations. We identified and validated three lead compounds and their intermediates using biochemical and cell-based assays. Fluorescence-based and HPLC-based enzymatic assays demonstrated potent inhibition of both the deacetylase and deacylase activities of HDAC11, with Inhibitor 6 and Inhibitor 3 exhibiting the strongest effects among the six compounds tested. Further, a decrease in lipid accumulation, reduced stability of the HDAC11 substrate SHMT2, as determined by immunoblot analysis and decreased cell viability, as assessed by MTT assay, confirmed HDAC11 inhibition in cellular models. The study shows that new HDAC11 inhibitors significantly reduce the viability of breast cancer cells and induce apoptosis; inhibitor 6, in particular, showed high potency, similar to the reference compound SIS-17. Flow cytometry showed that treated MDA-MB-231 cells exhibited cell-cycle arrest and increased apoptosis, a finding further confirmed by Annexin V/PI staining. Molecular analysis showed that BAX increased while BCL2 decreased, indicating that apoptotic pathways were activated in novel compound-treated MDA-MB-231 cells. The results suggest that inhibiting HDAC11 is an effective way to induce cancer cell death and provide a basis for further assessment of these compounds as potential treatments for breast cancer. Collectively, this study identifies novel zinc-chelating HDAC11 inhibitors containing a nitro-sp2 group, providing promising candidates for further therapeutic development.

Cholesterol is a key component of eukaryotic cell membranes, promoting membrane stability and modulating the function of many membrane proteins, including ion channels. In our previous work using purified human voltage-gated proton channel proteins, we showed that cholesterol inhibits the hHv1 channel by altering the conformational dynamics of its S4 segment, the key element that senses membrane voltage to control proton permeation. In the present work, we examined the effects of cholesterol analogs and potential sites in the hHv1 channel mediating cholesterol inhibition using site-directed mutagenesis and docking simulations. Our results showed that desmosterol, the immediate precursor of cholesterol, markedly attenuates cholesterol inhibition. Using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we showed that desmosterol attenuates cholesterol inhibition by promoting the intermediate and open state conformations of the S4 segment. Moreover, we identified multiple residues in the hHv1 channel that are critical for cholesterol inhibition, including Y141A in the S2 segment, which reduces cholesterol inhibition by nearly 3-fold. Our smFRET results showed that the Y141A mutation promotes the intermediate conformation in the S4 segment, which underlies the attenuation of cholesterol inhibition. Consistently, docking simulations also revealed multiple residues spanning the transmembrane domain, rather than clustered within a single localized pocket. Our work identified the key molecular determinant in the hHv1 channel that mediates cholesterol inhibition and also provided a mechanism linking the conversion between demosterol and cholesterol by DHCR24 to pH homeostasis in many cells, such as phagocytes, cardiomyocytes, neurons and microglial cells.

This paper presents and critically reviews the results of original and some literature based experimental studies conducted by the authors last years on the structural organization of DNA in dormant (starvation stress), anabiotic dormant (4 HR treatment) E. coli cells, as well as the K12 {Delta}dps strain, which lacks the Dps protein (Dps null E. coli). The experimental data includes small-angle synchrotron radiation diffraction (SAXS) and transmission electron microscopy (TEM) data. Synchrotron radiation diffraction experiments on K12{Delta}dps cells allowed us to conclude that peaks at 44.3, 22.1, and 14.8 angstrom resolutions are associated exclusively with ordered DNA organization. Peaks at 44.3, 22.1, and 14.8 angstrom resolutions are also observed for samples of dormant (starvation stress) cells and anabiotically dormant cells. Therefore, this ordered DNA organization also applies to samples of dormant and anabiotically dormant cells. A model is proposed that considers the ordered DNA organization in the cell as a cholesteric liquid crystal. The powder diffraction pattern calculated based on this model is compared with experimental small angle X ray scattering (SAXS) data obtained on Dps-null cell samples. The model completely reproduces the key features of the experimental diffraction pattern from Dps-null cell samples. Accordingly, the cholesteric liquid crystal model corresponds to DNA packaging in dormant and anabiotically dormant cells. Cholesteric liquid crystal ordering should be further considered in all models of cellular DNA packaging. To address the question of which structural organization of DNA predominates in the cell: the cholesteric liquid crystal or nanocrystalline or whether they coexist and fully manifest themselves under different external conditions, it is necessary to utilize the latest methodological advances in structural analysis.

Eukaryotic translation initiation is tightly regulated by interactions among translation initiation factors (eIFs) that ensure accurate start codon selection. The translation regulator, eIF5 mimic protein 1 (5MP1) contributes to this process by competing with eIF5 for binding to eIF2, thereby increasing the stringency of translation initiation. Despite its important regulatory role and emerging involvement in tumorigenesis, structural information on human 5MP1 remains limited. Here, we report the near-complete backbone and partial side-chain NMR resonance assignments of the C-terminal domain of human 5MP1 (residues 250-419), carrying a W404E substitution that disrupts dimerization. The WT protein forms a dimer at NMR concentrations, which increases the effective size of the protein and also causes disappearance of peaks corresponding to aminoacids at the dimer interface due to conformational exchange. Backbone resonance assignments were completed for 96.4% of the non-proline residues. Secondary structure was analyzed using Chemical Shift Index (CSI) and compared with the AlphaFold structural model. Regions of disagreement between the experimental and computational secondary structure assignments were further examined using 15N-NOESY-HSQC spectra, allowing experimental validation of local structural features. While the AlphaFold model accurately reproduces the overall fold of the 5MP1 C-terminal domain, several localized discrepancies were identified, particularly near the N- and C-terminal regions of the domain, where experimental NMR data support alternative secondary structure assignments. These resonance assignments and experimentally validated structural features provide a foundation for future investigations of the molecular interactions, dynamics, and functions of 5MP1 in translation initiation.

The High Mobility Group Box 1 (HMGB1) protein performs multiple essential functions in the body, ranging from DNA regulation to the activation and mediation of immune responses. However, HMGB1 has been also implicated in several pathological conditions, such as rheumatoid arthritis, sepsis, autoimmune diseases, tumors, and Alzheimer's disease. As a result, HMGB1 is of increasing interest as a therapeutic target. Binding to heparin has been reported to inhibit HMGB1's pathological activity during sepsis in clinical settings. In this work, we compare the interactions of HMGB1 with heparin and its' synthetic analog linear polyglycerol sulfate (lPGS) from the viewpoint of stability and changes to association behavior. This analysis focuses on thermal stability, secondary-structure changes, and particle-size evolution using nano-differential scanning fluorimetry (nanoDSF), circular dichroism spectroscopy (CD), and dynamic light scattering (DLS).

We present a simple and efficient way to add cis dialdehydes, phosphoryl groups, or cis diols to the 3 prime end of oligonucleotides using periodate oxidized nucleotides (oNTPs) and terminal deoxynucleotidyl transferase (TdT). The 3 prime end cis dialdehyde-modified oligos are generated by incubating TdT with an oligo for several minutes followed by addition of a oNTP and incubated at 30 degrees C for 30 minutes to an hour. After allowing the addition of the cis dialdehydes, heating the reaction mixture at 90 to 95 degrees C for 10 minutes yields oligonucleotides with 3 prime phosphoryl groups. The 3 prime cis diol modified oligos are synthesized by starting with 3 prime cis diol nucleotides (HO-NTPs). The cis dialdehyde modified oligonucleotides and cis diols may then be used for a variety of investigations such as studying the interaction of proteins with the 3 prime end of DNA, and possibly RNA. As an example, we demonstrate the efficacy of using an oligonucleotide modified with oGMP at the 3 prime end as an affinity label for TdT and identified a peptide fragment that has been shown to contain two of three aspartate residues found to be in the TdT active site.

The emergence of antibiotic-resistant pathogens such as Staphylococcus aureus demands accelerated antimicrobial discovery strategies. Artificial intelligence (AI) enables large-scale inference of candidate antimicrobial peptides (AMPs), yet experimental validation remains essential to determine whether predictions translate into biological function. Genome-guided mining, rather than unconstrained or randomly generated sequence exploration, offers a biologically grounded search space derived from organisms shaped by ecological and evolutionary pressures. Here, we evaluate this principle using Malassezia furfur, a skin-associated yeast that coexists with bacterial colonizers such as S. aureus, as a genomic source for AI-prioritized antimicrobial candidates. Candidate fragments were generated from two M. furfur genomes, filtered by physicochemical properties, prioritized with deep-learning AMP predictors, synthesized, and experimentally characterized. Selected peptides underwent cross-kingdom antimicrobial screening against S. aureus, combining kinetic growth and ultrastructural assays, complemented by in silico structural prediction, lipid-membrane interaction analysis, and human keratinocyte cytotoxicity evaluation. AI-guided genomic mining enriched biologically motivated sequence space for peptides with measurable antimicrobial activity, while revealing biases and generalizability limits of AI-based AMP inference. Closing the loop between genome-derived candidate generation, AI-based inference, synthesis, and functional characterization, this study provides an experimental assessment of model-guided AMP discovery and a reproducible route from computational prediction to validated antimicrobial candidates.

Long noncoding RNAs encode for microproteins that regulate cellular functions. Small regulatory peptide of amino acid response (SPAR) is a microprotein in the lysosome that responds to amino acid availability of the cell. In this study, we investigated the interactions between SPAR and SLC38A9, a lysosomal amino acid transporter and receptor involved in the mechanistic target of rapamycin 1 (mTORC1) pathway. We found that SPAR binds SLC38A9 and inhibits arginine transport in SLC38A9. Moreover, the downstream recruitment of Rag GTPases is also inhibited when SPAR is present in SLC38A9 liposomes. Docking model shows potential interactions between SPAR and SLC38A9. Together, these findings reveal the mechanism of mTORC1 inhibition through microprotein SPAR and illustrates the power of non long coding RNAs in altering cellular functions. Statement of SignificanceMicroproteins encoded from long noncoding RNAs are emerging as critical regulators of many pathways. This study investigates a novel mechanism of SPAR microprotein that directly regulates the mechanistic target of rapamycin complex1 (mTORC1) signaling pathway through the lysosomal amino acid transporter SLC38A9. SPAR blocks both arginine transport and the downstream recruitment of Rag GTPases. These findings provide critical results in how SPAR controls cellular amino acid availability, while broadly highlighting the powerful regulatory mechanism of microproteins in cellular processes.

Antisense oligonucleotides (ASO) are nucleotide polymers that hybridize to sense strands and have been successful in treating a variety of diseases. A wide range of strategies have been investigated to optimize and develop ASO for clinical studies. A key objective for this study was to provide an overview of the range of detailed data that get be obtained and provide an updated method review on how to design surface plasmon resonance (SPR) kinetic experiments for DNA oligonucleotide hybridization studies that can also be applied to other ASO including peptide nucleic acids (PNA). We describe many lessons learned from published literature and provide a state-of-the-art strategy and methods for generating not only kinetic but also thermodynamic characterizations of oligonucleotide hybridization. In this study we have performed an SPR kinetic and thermodynamic analysis for the hybridization of HIF1 antisense DNA strands to its immobilized Intron2-Exon3 splice site sense DNA strand to provide insight, in general, on the optimal length and insight into optimal design of DNA ASOs. We provide a process on how to design experiments to: 1.) obtain oligonucleotide-length dependent kinetics, 2.) analyze reactions to obtain association and dissociation rate kinetics (ka, kd), assess if hybridization follows a 2-state model and to obtain kinetic dissociation constants (Kd), 3.) perform temperature-dependent hybridization kinetics to obtain thermodynamic values ({Delta}H{degrees}, {Delta}S{degrees} and {Delta}G{degrees}) that can give insight into the molecular interactions driving hybridization, 4.) compare experimental thermodynamic values to values derived from nearest-neighbor prediction models to identify atypical reactions and importantly 5.) enable calculations to predict oligomer hybridization affinity at the physiological 37 {degrees}C temperature to asses if the design of the oligomer will have the required cellular activity for a therapeutic effect. The strategy and results presented throughout the paper are compared to previous SPR reports and suggestions made to optimize kinetic studies.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by progressive motor neuron degeneration. Mutations in the SOD1 gene represent the second most common genetic cause of ALS (ALS), and distinct SOD1 missense variants present with markedly different clinical profiles. A4V leads to an aggressive form of the disease (median survival [~]1y), H46R confers a mild, slowly progressive course and I113T exhibits an intermediate phenotype. The molecular basis by which these mutations produce divergent clinical outcomes remains poorly understood. We performed extensive classical molecular dynamics simulations of wild-type SOD1 and the three ALS-associated variants in the apo monomeric state to attempt to investigate the mechanisms behind such phenotypic differences. Structural stability, global compactness, and conformational flexibility, as well as analysis of collective motions between residues and estimation of free energy, were assessed. The H46R, A4V, and I113T variants exhibited distinct dynamic behaviours, highlighting differences in structural stability, local flexibility, and intramolecular interactions. These findings suggest that specific structural regions may contribute differently to protein dysfunction and could represent key elements for understanding the relationship between molecular dynamic properties and the differing clinical severity associated with these variants. Most strikingly, H46R exhibited exceptional structural stability across every analytical level, the lowest global deviation, most attenuated local flexibility, strongest internal dynamic coordination, and the deepest, most confined free energy basins of any system examined. This convergent multi-layered evidence of structural restraint provides a compelling mechanistic basis for the mild and slowly progressive clinical course of H46R ALS, suggesting that enhanced conformational rigidity, rather than bulk destabilisation, is the defining biophysical feature of this variant, and that its pathogenic mechanism operates through a route fundamentally decoupled from the aggregation-driven toxicity that characterises the more aggressive SOD1-ALS mutations.

The classical understanding of allostery was initially grounded in two-state models, such as MWC and KNF, where structure and function are inextricably linked through transitions between low-(T) and high-affinity (R) states. Here, we show Yeast chorismate mutase (CM) provides a vivid example of the growing list of exceptions to the traditional T vs R two-state allosteric paradigm. While CM exhibits dynamic sampling of the R-state in the presence of the activator tryptophan (Trp), suggesting a conformational selection (CS) mechanism, we present multiple instances where conformational status and catalytic activity are decoupled. Using NMR spectroscopy and kinetic assays, we identify CM variants that reside almost exclusively in the T conformation can exhibit maximal activity, while others that predominantly occupy the R conformation are weakly active. Quantitative comparison of experimental data with a parameterized CS model reveals deviations of up to two orders of magnitude, ruling out the simplest two-state model for substrate affinity modulation in this system. We propose that the observed T-to-R switching in CM is "incidental", a byproduct of an evolved energy landscape that allows access to the substrate-bound pose but does not mechanistically determine affinity. Our findings suggest that allosteric regulation in CM may instead be driven by local features of the ground-state ensemble, which operate independently of global T/R status. This work further highlights an emerging view that the mere observation of a pre-sampled active conformation does not sufficiently prove a two-state mechanism and further underscores the need for deeper ensemble-based perspectives in protein engineering and allostery.

The rotary motor F1-ATPase has been extensively studied as a model molecular machine, yet rational engineering of its catalytic activity remains challenging because ATP hydrolysis is regulated by long-range intersubunit allostery and large conformational transitions. Here, we developed a homolog-guided engineering strategy to increase the maximum rotation rate of the thermophilic Bacillus PS3 F1-ATPase (TF1). Candidate mutation sites were first identified by comparing TF1 with the homologous enzymes bovine mitochondrial F1 (bMF1) and Paracoccus denitrificans F1 (PdF1), both of which exhibit higher maximum rotation rates than TF1. Systematic exploration of these sites identified four activity-enhancing hotspots, followed by focused hotspot exploration and machine-learning-assisted prioritization of combinatorial mutants. The best mutant, TF1({beta}Y313L/{beta}E332S), exhibited a 1.8-fold higher maximum rotation rate than TF1(WT) while retaining its functional thermostability. Interestingly, activity-enhancing substitutions were not limited to the residues conserved in both bMF1 and PdF1, indicating that the bMF1-PdF1 consensus substitutions effectively identify activity-enhancing hotspots rather than uniquely defining the optimal amino acid. Machine-learning-assisted exploration efficiently prioritized highly active mutants, although the predictive performance was limited by the relatively small training dataset and epistatic interactions among mutations. Kinetic and structural comparisons further provided mechanistic insights into the enhanced catalytic activity of the engineered mutant. Together, these results establish a practical strategy for engineering complex molecular motors by combining homolog-guided hotspot identification with focused hotspot exploration.

Bacterial sortase enzymes are cysteine transpeptidases at the surface of Gram-positive bacteria that ligate substrates to the cell wall. In addition, these enzymes are powerful tools in protein engineering applications via sortase-mediated ligation (SML) due to their covalent attachment of two substrates, with one containing a pentapeptide recognition motif with sequence LPXTG, where X=any amino acid, and the second, an N-terminal glycine. The class A sortase from Staphylococcus aureus (saSrtA) was the first to be identified, and over 25 years later, the most widely used SML variants continue to be derivatives of a directed-evolution-identified pentamutant of saSrtA, or saSrtA5M. We previously characterized P94, a position mutated in saSrtA5M that interacts directly with a structurally conserved loop (the {beta}7-{beta}8 loop) near the active site of wild-type saSrtA only in the inactive conformation. This work revealed that the single P94X mutation dramatically affects relative saSrtA activity, as well as specificity for the P2 (or X) position in the LPXTG recognition motif. This is largely driven by Km effects. Here, we further interrogated P94 by probing structural changes in the active, apo state of saSrtA in the presence of the P94D mutation, as well as via mutations in Y187, the {beta}7-{beta}8 loop residue hypothesized to interact directly with P94. The saSrtA enzyme is allosterically activated by calcium; therefore, we were interested if P94D would induce structural changes in the calcium-bound apo enzyme. We used 1H-15N NMR experiments to compare spectra between enzymatically inactive variants of saSrtA with and without the P94D mutation. We also used NMR to calculate relative binding affinities for a pentapeptide substrate to these variants, as well as enzymatically inactive saSrtA5M. Our NMR data, in combination with enzymatic assays using active variants confirmed differences in the active, apo states of these enzymes. Overall, this work provides additional atomic detail regarding the importance of the P94 residue in saSrtA substrate recognition.

Protein phosphatase 2A containing the B56{delta} regulatory subunit (PP2A-B56{delta}) is a critical signaling enzyme whose dysregulation is associated with cancer, neurodegenerative disorders, and Jordan's syndrome, a severe intellectual disability disorder caused by mutations in B56{delta}. Unlike other PP2A holoenzymes, PP2A-B56{delta} is regulated through a unique dual autoinhibition mechanism in which the N- and C-arms occlude the catalytic site while a substrate-mimicking short linear motif (SLiM) blocks the substrate-binding pocket. Although disease-associated mutations have been shown to alter enzyme activity, the molecular mechanism underlying activation of PP2A-B56{delta} and the effects of pathogenic mutations remain poorly understood. Here, we combined cryo-electron microscopy (cryo-EM), enhanced-sampling molecular dynamics (MD) simulations, Markov state model (MSM) construction, and transition-state analysis using Transition State identification via Dispersion and vAriational principle Regularized neural networks (TS-DAR) to characterize the conformational landscape of the disease variant E198K. Our cryo-EM analysis identified two distinct structures of E198K: an inactive closed-form with the N/C-arms resolved and an active loose-form in which the N/C-arms become highly flexible and could not be fully resolved. These structures therefore established that activation is governed by conformational changes of the N/C-arms but did not reveal the underlying mechanism. Starting from the inactive closed-form, we generated over 1,600 trajectories with an average length of 1,260 ns combined for E198K and wild-type (WT) PP2A-B56{delta}. TS-DAR identified four metastable states and two major activation pathways connecting inactive and active conformations. We found that activation occurs through progressive loosening of the N/C-arm interface while maintaining the overall holoenzyme architecture, rather than a complete opening of the interface. This mechanism exposes both the catalytic site and substrate-binding pocket. Comparison of E198K and WT revealed that the disease-associated mutation shifts the conformational equilibrium toward active states while leaving the transition-state ensemble largely unchanged. Mechanistically, E198K disrupts a salt-bridge network and weakens interactions between the internal loop and the C-arm that normally stabilize active-site occlusion. The resulting increase in C-arm mobility promotes active-site exposure and explains the elevated catalytic activity of the mutant. Together, these findings establish a previously uncharacterized activation mechanism for PP2A-B56{delta} and provide an atomic-level explanation for how the pathogenic E198K mutation allosterically promotes holoenzyme activation.

We present the solution structures of HIV-1 proteins NC(p7)1-55 corresponding to the full-length NC(p7) and mature p6. The studies were carried in water and, to mimic the membrane, in micellar DPC (Dodecylphosphocholine) conditions. Our results unravel for the first time the structure adopted by the N-terminal amino acids of the free NC(p7)1-55, with the formation of a small helix spanning residues F6 to R10. Our NMR and Fluorescence Anisotropy data disclose an interaction between NC(p7)1-55 and p6 both in water and DPC, with respective Kd of 2.5mM and 370 mM at 23{degrees}C. The interaction is thus strengthened in lipidic conditions. Protein p6 stabilizes the N-terminus of NC(p7)1-55 while increasing at the same time the dynamic of the first zinc finger. Although the entire p6 sequence is involved in the interaction, we show that its C-terminal region is particularly sensitive to the presence of NC(p7)1-55, with a propensity of forming a a helix ranging from amino acids S111 to F116. This study brings experimental evidence of a direct protein-protein interaction between p6 and the N-terminal region of NC(p7)1-55. We further show that such interaction is readily accommodated within the NC(p15) framework and hypothesize that it may facilitate the selective assembly of assembly of the viral genomic RNA (gRNA) in the cell.

The rising incidence of invasive fungal diseases emphasizes the need for novel therapeutic strategies, including membrane-targeting antifungal agents, which require representative lipid models for detailed molecular-level studies. In this work, we propose a consensus quinary fungal plasma membrane model based on lipidomic literature data, specifically PC:PE:PI:PA:PS phospholipid model with ratio of 44:29:13:8:6. Using a bottom-up approach, we characterized the biophysical properties of this system - with particular emphasis on mechanical parameters such as bending rigidity and area compressibility - by combining molecular dynamics simulations with experimental flicker-noise and ATR-FTIR spectroscopies. Furthermore, we investigated the effect of two key non-phospholipid components: ergosterol and triacylglycerols. Biophysical analysis revealed that DPPI and its specific interactions with DSPS induced the most substantial deviations in baseline membrane parameters, particularly area per lipid, membrane thickness, and area compressibility, while DSPS influenced bending rigidity change and DLiPA primarily affected lipid packing defects. In addition, ergosterol and TGs were found to influence all of the investigated parameters to different degree. Notably, the overall biophysical profile of the proposed FPMM closely mimicked that of natural vesicles derived from yeast lipid extracts, establishing this model may provide a reliable platform for studying fungal membrane biophysics and lipid-targeting interactions.

The binding affinities of a chimeric analog of a riboflavin derivative linked to biotin, (6- (7,8-dimethyl-2,4-dioxo-3,4-dihydrobenzo[g]pteridin-10(2H)-yl)hexyl 5-((3aS,4S,6aR)-2- oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate), referred to as C6-Rf-biotin-tag, to the riboflavin binding retain or streptavidin are in the M range, 1.29 {+/-} 0.277 and 3.00 {+/-} 0.459, respectively. These values suggest that C6-Rf-biotin-tag has potential applications in diagnostic assay and labelling target flavin binding proteins. The C6-Rf-biotin-tag which was characterized with respect to physical and biochemical properties retains UV/Vis spectroscopic and fluorescence behavior similar to riboflavin.

This study proposes a novel therapeutic strategy to suppress cancer growth by modulating the MDM2-p53 axis via Alternative Polyadenylation (APA). MDM2 normally promotes tumorigenesis by ubiquitinating and degrading the tumor suppressor p53. In cancer cells, preferential use of proximal polyadenylation signals (PAS) results in shortened 3'UTRs, allowing oncogenic transcripts like MDM2 to evade nuclear sequestration mediated by Inverted Alu (IRAlu) double-stranded RNA structures. We hypothesized that forcing distal PAS usage would elongate the MDM2 mRNA, promoting its nuclear retention and reducing protein translation, thereby restoring p53 activity. Using CRISPR-Cas9, we targeted and deleted the most frequent proximal PAS in the MDM2 3'UTR of A549 cells. Successful genome editing was confirmed via PCR. As expected, Western blot analysis showed a significant reduction in MDM2 expression in PAS-edited cells. However, experimental outcomes contradicted our initial hypothesis: edited cells exhibited higher viability under doxorubicin treatment compared to wild-type cells. Furthermore, despite decreased MDM2 levels, a concurrent reduction in phosphorylated p53 (p-p53) was observed. These unexpected results suggest that MDM2 3'UTR elongation may trigger a non-canonical regulatory mechanism that bypasses the traditional MDM2-p53 interaction. This study highlights the complexity of post-transcriptional regulation and suggests that APA-mediated gene modulation can induce unforeseen compensatory survival pathways in cancer cells, necessitating further investigation into the broader functional landscape of elongated 3'UTRs.

Mucopolysaccharidosis IIIC (Sanfilippo syndrome type C) is a rare lysosomal storage disorder caused by loss-of-function mutations in HGSNAT, which encodes an enzyme involved in heparan sulfate (HS) degradation, leading to impaired HS catabolism, lysosomal accumulation, and progressive neurodegeneration. Because enzyme replacement therapies have limited penetration across the blood-brain barrier, substrate-reduction therapy represents an alternative therapeutic strategy. Here, N-deacetylase/N-sulfotransferase 1 (NDST1), a key enzyme responsible for HS biosynthesis, was investigated as a potential substrate-reduction target. A structure-based computational pipeline was used to identify and evaluate inhibitors targeting the NDST1 sulfotransferase domain. Approximately 4.1 million drug-like compounds and FDA-approved drugs were screened by molecular docking, followed by pharmacokinetic filtering, molecular dynamics simulations, and MM/PBSA binding free energy calculations. In parallel, peptide binders targeting the same site were generated using diffusion-based protein design and evaluated using molecular dynamics and MM/GBSA analysis. Four chemically distinct small-molecule scaffolds and three peptide candidates were identified as stable binders to the NDST1 active site. The lead small-molecule candidate exhibited a predicted binding free energy of -13.36 {+/-} 5.87 kcal mol-1. These provide a focused set of candidates for further investigation and support the feasibility of targeting NDST1 as a substrate-reduction strategy for MPS IIIC.