Unique molecular architecture of N-glycosylated TM4SF5 dimer highlights evolutionary and structural divergence among small four-transmembrane protein families.

Background/Objectives: Breast cancer is the most prevalent malignancy among women, and paclitaxel (PTX) is a first-line chemotherapeutic, but chemoresistance driven by ATP-binding cassette (ABC) transporters limits its efficacy. Single-target ABC inhibitors fail due to toxicity and cooperative transporter activity, creating an urgent need for safe multi-target strategies. Membrane cholesterol-rich lipid rafts support ABC transporter function, making cholesterol a key chemoresistance target. This study explored a cholesterol-targeted approach for overcoming PTX resistance. Methods: A PTX-resistant breast cancer line (MCF-7/PTX) showing ABCB1/ABCC3 co-upregulation and enriched cholesterol rafts was established. The effects of Polyphyllin H (PPH), a steroidal saponin from Paris polyphylla, were compared with lovastatin, a biosynthetic cholesterol inhibitor. In vitro and in vivo assays investigated Polyphyllin H’s cholesterol binding and effects on transporters, PTX accumulation, and tumor growth. Results: PPH directly binds membrane cholesterol, disrupting lipid rafts, downregulating ABCB1/ABCC3, reducing drug efflux, and increasing intracellular PTX to restore sensitivity. PPH showed superior cholesterol-binding and resistance-reversal efficacy than lovastatin, with faster, stronger PTX-enhanced cytotoxicity and tumor suppression. Conclusions: PPH reverses PTX resistance by targeting cholesterol-lipid rafts to inhibit multiple ABC transporters. This offers a safer adjuvant for PTX-based breast cancer therapy and a translational framework for other drug-resistant malignancies.

An automated platform for lentiviral transduction for HSC gene therapy promotes fitness of hematopoietic stem cells combined with higher transduction efficiency

Structural insights of stomatin from native human platelets by cryo-EM coupled with “Build and Retrieve” (BaR) data processing.

Structural insight into GPR155-mediated cholesterol sensing and signal transduction.

The existence of linear cholesterol-recognition motifs in transmembrane domains has long been debated. Evolutionary molecular dynamics (Evo-MD) simulations—genetic algorithms guided by (coarse-grained) molecular force-fields–reveal that thermodynamic optimal cholesterol attraction in isolated alpha-helical transmembrane domains occurs when multiple consecutive lysine/arginine residues flank a short hydrophobic segment. These findings are supported by atomistic simulations and solid-state NMR experiments. Our analyses illustrate that linear motifs in transmembrane domains exhibit weak binding affinity for cholesterol, characterized by sub-microsecond residence times, challenging the predictive value of linear CRAC/CARC motifs for cholesterol binding. Membrane protein database analyses suggest even weaker affinity for native linear motifs, whereas live cell assays demonstrate that optimizing cholesterol binding restricts transmembrane domains to the endoplasmic reticulum post-translationally. In summary, these findings contribute to our understanding of cholesterol-protein interactions and offer insight into the mechanisms of protein-mediated cholesterol regulation within membranes.

Bast fibrous plants are recognized as potential sources of pectin, yet the structural characteristics and functional properties of pectic polysaccharides derived from these plants remain insufficiently investigated. In this study, three pectic polysaccharides (HP, KP, and RP) were isolated from the bast fibers of Cannabis sativa L. (hemp), Hibiscus cannabinus L. (kenaf), and Boehmeria nivea L. (ramie) using an ammonium oxalate solution. Their chemical composition, monosaccharide profile, molecular weight, microstructure, and functional groups were analyzed by spectroscopic and chromatographic techniques. These polysaccharides exhibited remarkable uronic acid content (50.49–61.14%), a relatively low degree of esterification (19.51–34.26%), and high molecular weights ranging from 147.10 to 242.16 kDa. The predominant neutral sugars identified were rhamnose, arabinose, and galactose. Furthermore, HP, KP, and RP demonstrated superior thermal stability, emulsifying properties, water/oil holding capacity, and cholesterol binding ability compared with commercial citrus pectin (CP). They also exhibited stronger radical scavenging activity, with KP showing particularly notable antioxidant performance (IC50 = 1856 and 1485 μg/mL for the DPPH and ABTS assays, respectively). Overall, these findings indicate that bast fibrous plants are promising alternative sources of pectic polysaccharides with favorable antioxidant properties, supporting their potential application as food additives or functional ingredients.

Synthesis, characterization, and in vitro hypolipidemic mechanisms of sugar-SA triazole conjugates as potent pancreatic lipase inhibitors.

302 Role of cholesterol on CFTR single channel characteristics and potential cholesterol binding domains

Recognition of Specific PIP2-subtype Composition Triggers the Allosteric Control Mechanism for Selective Membrane Targeting of Cargo Loading and Release Functions of the Intracellular Sterol Transporter StarD4.

The proposed physiological roles of bile acids have expanded beyond the digestion of fats to encompass cell signaling via the activation of a variety of nuclear and plasma membrane receptors in multiple organ systems. The current in silico study was inspired by previous observations from our group and others that bile acids interact functionally with cardiac, pulmonary, and gastrointestinal muscarinic receptors and more recent work demonstrating allosteric binding of cholesterol, the parent molecule for bile acid synthesis, to M1 muscarinic receptors (M1R). Here, we computationally tested the hypothesis that bile acids can allosterically bind to M1R and thereby modulate receptor activation. Utilizing de novo site identification by the ligand competitive saturation (SILCS) method, putative novel allosteric binding sites of bile acid targeting M1R were identified. Molecular dynamics simulations were used to uncover the molecular details of the activation mechanism of M1R due to agonist binding along with allosteric modulation of bile acids on M1R activation. Allosteric binding of bile acids and their glycine and taurine conjugates to M1R negatively impacts the activation process, findings consistent with recent reports that M1R expression and activation inhibit colon cancer cell proliferation. Thus, bile acids may augment colon cancer risk by inhibiting the tumor suppressor actions of M1R. When validated experimentally, these findings are anticipated to shed light on our understanding of how bile acids in the membrane microenvironment can allosterically modulate the function of M1R and possibly other G protein-coupled receptors.

The steroidogenic acute regulatory protein (StAR) governs the rate-limiting step of steroid hormone biosynthesis by facilitating cholesterol transfer from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM). This essential function initiates pregnenolone synthesis by P450 family 11 subfamily A member 1 (CYP11A1, cytochrome P450scc) within IMM. Beyond its biochemical role, StAR is a critical developmental protein, with spatiotemporally restricted expression during fetal adrenal and gonadal differentiation. Its activity is tightly regulated at multiple levels, including transcriptional control by transcription factors, GATA post-translational phosphorylation, mitochondrial targeting, and proteolytic degradation. Structurally, StAR functions through a dynamic molten globule-like conformation and a conserved StAR-related lipid transfer (START) domain that mediates cholesterol binding. StAR interacts with mitochondrial proteins such as nonselective voltage-gated ion channel VDAC (VDAC), translocator protein (TSPO), and ATPase family AAA domain-containing protein 3A (ATAD3A), forming part of the transduceosome complex that coordinates cholesterol transfer. Mutations in STAR, particularly within the START domain), cause lipoid congenital adrenal hyperplasia (CAH), a disorder marked by impaired steroidogenesis and disrupted endocrine organ development. This review integrates current knowledge on the molecular and developmental roles of STAR, emphasizing how its precise regulation is essential for embryonic steroidogenesis. Understanding StAR's function at the interface of lipid transport and organogenesis provides critical insight into congenital steroidogenic disorders and potential avenues for therapeutic intervention.

Cholesterol differentially modulates the activity of opioid and muscarinic receptors via a common binding site.

The ion channel-forming natural product amphotericin B (AmB) can serve as a molecular prosthetic for the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel and thereby restore host defenses in cultured cystic fibrosis (CF) airway epithelia. This is despite the fact that the permeability of AmB-based channels favors cations, and these channels lose their capacity to increase airway surface liquid (ASL) pH in CF airway epithelia at high concentrations. We hypothesize that modifying such channels to favor anion permeability would make them more CFTR-like and thus increase their potential therapeutic effects compared to AmB. Here we show that a synthetic derivative of AmB, AmB-AA, which has an added positively charged appendage and forms ion channels with an improved relative permeability to anions, outperformed AmB in increasing the ASL pH in CF airway epithelia at both low and high concentrations. Further modifications led to another AmB derivative, C2’epiAmB-AA, that also minimized cholesterol binding and thus toxicity to cultured CF airway epithelia and was an effective surrogate for CFTR in primary cultured airway epithelia from people with CF.

Black sticky rice tapai is a traditional Indonesian fermented food that has long been consumed by the public for its distinctive taste and high cultural value. However, with the increasing demand for functional foods and natural-based products that can support health, attention for black sticky rice tapai has shifted from being simply a traditional food to an object of scientific study in the nutraceutical field. Black sticky rice, the basic ingredient for tapai, contains important bioactive compounds such as anthocyanins, flavonoids, B-complex vitamins, and soluble fiber, which have been shown to play a role in lowering cholesterol levels, inhibiting LDL cholesterol oxidation, and increasing HDL cholesterol levels in the blood. The fermentation process further enhances the biological activity of these compounds, making black sticky rice tapai have a higher therapeutic value than ordinary black sticky rice. The purpose of this study was to examine the potential of black sticky rice tapai as a natural, tradition-based solution for lowering cholesterol levels, while also supporting the development of local foods into modern health products that are safe, effective, and affordable. The study was conducted using a descriptive literature review method of various scientific articles, textbooks, and relevant research results that discuss the nutritional content of black sticky rice, the benefits of fermentation, and the effect of active components on lipid metabolism. The study results show that regular consumption of black glutinous rice tapai has the potential to lower total and LDL cholesterol levels and increase HDL through a combination of antioxidant effects, cholesterol binding by fiber, and increased fat excretion. In addition, the potential of black glutinous rice tapai as a nutraceutical is also relevant in supporting the preservation of local-based culture and food independence.

Microalgae, with their high photosynthetic efficiency and sustainability, hold promise to produce bioactive compounds, chemicals, cosmetics, and biofuels. This study aims to understand the molecular mechanisms of bioactive compounds from microalgae using integrative bioinformatics approaches to identify their potential therapeutic applications. Gene expression profiles from the GSE113144 and GSE115827 datasets were retrieved from the GEO database using keywords such as liver disease, microalgae, and bioactive compounds. Different expressed genes (DEGs) were identified using the GEO2R tool. Subsequently, a PPI network was constructed to identify hub genes and key regulatory elements. The findings were further cross-validated using a range of bioinformatics tools, databases, and literature to explore their potential applications in drug development, nutraceuticals, and disease modulation. Following oxo-fatty acid treatment, 2051 differentially expressed genes (DEGs) were identified, while 399 DEGs were detected after sea spray aerosol treatment, with 39 genes shared between the two treatments. These DEGs were primarily enriched in immune and metabolic processes. Protein-protein interaction analysis revealed ten key hub genes: PBK, CENPA, ASPM, DLGAP5, DEPDC1, SPC25, CDCA3, HJURP, ERCC6L, and KIF18B, which are involved in immune and metabolic responses. Functional enrichment highlighted roles in cholesterol and fatty-acyl-CoA binding, peptidoglycan recognition, metal ion binding, and protease activity. Notably, PBK and CDCA3 are associated with approved drugs, suggesting potential for therapeutic repurposing. The molecular functions enriched among hub genes, such as cholesterol binding, fatty-acyl-CoA binding, peptidoglycan receptor activity, and metal ion binding, suggest actionable pathways that could be pharmacologically modulated. These targets are highly relevant to diseases such as NAFLD and chronic inflammation. The identification of druggable hub genes and enriched immune-metabolic functions provides a foundation for further preclinical and translational research. This study offers valuable insights into the molecular mechanisms underlying human immune and metabolic responses to sea spray aerosols and oxo-fatty acids, identifying cellular pathways and processes that are often regulated in human immune and metabolic responses to various microalgae. Overall, this study enhances our understanding of the potential therapeutic applications of microalgae-derived bioactive compounds, offering potential breakthroughs in drug discovery and nutraceutical development.

The lysosomal cholesterol sensor LYCHOS regulates mTORC1 signaling by coupling cholesterol sensing to GATOR1-Rag GTPase modulation, yet its structural mechanisms remain unclear. Here we report six cryo-electron microscopy structures of human LYCHOS, depicting five distinct states. These are categorized into a contracted state when complexed with a sufficient amount of the cholesterol analogue cholesteryl hemisuccinate (CHS), and an expanded state when CHS is deficient. The structure forms a homodimer, within each monomer the transmembrane region is divided into a permease-like domain (PLD) and a GPCR-like domain (GLD) with two clearly defined adjacent cholesterol binding sites between them. Cholesterol binding induces a translation of GLD towards PLD and exposes the cytosolic extension of transmembrane 15, which interacts with GATOR1. Our results elucidate the structural mechanism of cholesterol sensing by the mTORC1 pathway, providing a structural basis for developing inhibitors that selectively target mTORC1 pathway by blocking LYCHOS in its expanded state.

Modifying the surface chemistry of poly-(3,4-ethylenedioxythiophene) (PEDOT) with biological moieties is of interest for optimizing interactions in biointerfacing applications. Here, we demonstrate the attachment of cholesterol, a lipid prevalent in animal blood and cell membranes, and cysteine, an amino acid prevalent in proteins, onto PEDOT surfaces through efficient thiol-maleimide click-chemistry between maleimide-functionalized PEDOT (PEDOT-MA) and corresponding thiol derivatives. The reaction process of cholesterol post-treatment was actively monitored by electrochemical impedance spectroscopy (EIS) taken at different incubation times. An equivalent circuit model was used to deconvolute the impedance data. It was found that the charge transfer capability, as well as the ion diffusion within the PEDOT film, decreased systematically with incubation time. However, the film resistance remained unchanged in this process, indicating the relatively unaffected bulk properties. This finding was supported by scanning electron microscopy (SEM) results where no significant morphological changes were observed after the post-treatment. In addition, the charge storage capability also decreased, with only 23% of the original capacitance remaining after the post-treatment. The water contact angle increased from 23 to 115° after the cholesterol binding, confirming the formation of a hydrophobic film that correlates with the decreased charge transport behavior. In contrast, the attachment of cysteine showed almost no effect on the charge transport behavior and charge storage capability of PEDOT-MA. Contact angle measurements confirmed the high hydrophilicity of the cysteine-treated film. Again, SEM experiments showed there were no significant changes in the surface morphology. X-ray photoelectron spectroscopy (XPS) confirmed the presence of cholesterol and cysteine on the sample surface. Our results demonstrate that thiol-maleimide click-chemistry provides an efficient and effective route for the surface functionalization of PEDOT with biomolecules.

Intracellular cholesterol homeostasis is regulated by the sterol response element-binding protein (SREBP) pathway through the SREBP cleavage-activating protein (SCAP) and the insulin-induced gene protein (INSIG). Previous studies highlighted that high concentrations of cholesterol mediated the dimerization between INSIG and SCAP, which led to an inhibition of cholesterol uptake and biogenesis. However, the molecular understanding of the SCAP-INSIG and cholesterol interactions remains elusive. Here, we used coarse-grained (CG) and atomistic (AT) molecular dynamics (MD) simulation to determine interactions between INSIG, SCAP, and cholesterol. Our work highlighted the novel binding pocket in the luminal domain at atomistic resolution. Using a combination of the AlphaFold3 model and Go̅-martini force field, we showed that loop 7 dynamics are crucial for cholesterol binding and are able to highlight conserved residues, which lead to INSIG-SCAP dimerization. Together, our work highlights novel mechanisms of cholesterol sensor pathways and will benefit the development of novel therapeutic strategies for diseases, such as neuroinflammatory disease, caused by irregular cholesterol homeostasis.

Corrigendum to “Diphenylpyraline derivative JKH10 behaves as a CB1 receptor agonist and docks at cholesterol and pregnenolone binding sites” [The Journal of Pharmacology and Experimental Therapeutics 389(3S), 2024