Search Results (319)

The function of pleckstrin homology (PH) domains is to recognize and bind to specific phosphoinositides in the membranes as part of diverse cellular signaling processes. The structure of some PH domains has been solved by X-ray crystallography, but structures of many PH domains remain to be elucidated. In green alga Chlamydomonas reinhardtii, none of the PH domains have been crystallized or characterized. The goal of our study was to model and characterize in detail the structures of all eleven of the PH domains identified in C. reinhardtii. Our computational strategy of integrating the information available on sequence, structure, and function with modeling and biophysical characterization has uncovered new biological predictions for these proteins. These predictions can be validated by future rationally designed experimental studies as an extension of this work. Our results suggest that nine of the eleven C. reinhardtii PH domains show the classical electrostatic polarization of PH domains with a positively charged binding pocket and negatively charged opposing end. Our docking results predict only two PH domains bind specifically to a particular phosphoinositide, while all the other nine PH domains may be able to bind various inositol phospholipids. The lack of preference for a specific phosphoinositide headgroup implies that the positive charge in the binding pocket of the PH domains may be crucial in driving the interaction with the negatively charged phosphoinositides in a non-specific or promiscuous manner. We identified putative homologs of Dynamin GTPase, calcium/calmodulin-dependent kinase, Arf GAP, Rhythm of Chloroplast 23 (ROC23), and oxysterol binding proteins in C. reinhardtii that contain PH domains. In addition, we identified two PH domain-containing proteins that may play a role in the mating process and others that may be important for signaling under phosphate deficiency. Full article

Oxidized phospholipids (OxPLs) are increasingly recognized as biologically active lipids involved in various pathologies. Both exposure to pathogenic factors and the efficacy of protective mechanisms are critical to disease development. In this study, we characterized an immunoassay that quantified the total capacity of the plasma to degrade or mask OxPLs, thereby preventing their interaction with cells and soluble proteins. OxLDL-coated plates were first incubated with human blood plasma or a control vehicle, followed by an ELISA using a monoclonal antibody specific to oxidized phosphatidylethanolamine. Pretreatment with the diluted blood plasma markedly inhibited mAb binding. The masking assay was optimized by evaluating the buffer composition, the compatibility with various anticoagulants, potential interfering compounds, the kinetic parameters, pre-analytical stability, statistical robustness, and intra- and inter-individual variability. We propose that this masking assay provides a simple immunological approach to assessing protective mechanisms against lipid peroxidation products. Establishing this robust and reproducible method is essential for conducting clinical association studies that explore masking activity as a potential biomarker of the predisposition to a broad range of lipid-peroxidation-related diseases. Full article

Phosphoinositide-binding pleckstrin homology (PH) domains interact with both phospholipids and proteins, often complicating their use as specific lipid biosensors. In this study, we introduced specific mutations into the phosphatidylinositol 3,4,5-trisphosphate (PIP₃)-specific PH domains of protein kinase B (Akt) and general receptor for phosphoinositides 1 (GRP1) that disrupt protein-mediated interactions while preserving lipid binding, in order to enhance biosensor specificity for PIP₃, and evaluated their impact on plasma membrane (PM) localization and lipid-tracking ability. Using bioluminescence resonance energy transfer (BRET) and confocal microscopy, we assessed the localization of PH domains in HEK293A cells under different conditions. While Akt-PH mutants showed minimal deviations from the wild type, GRP1-PH mutants exhibited significantly reduced PM localization both at baseline and after stimulation with epidermal growth factor (EGF), insulin, or vanadate. We further developed tandem mutant GRP1-PH domain constructs to enhance PM PIP₃ avidity. Additionally, our investigation into the influence of ADP ribosylation factor 6 (Arf6) activity on GRP1-PH-based biosensors revealed that while the wild-type sensors were Arf6- dependent, the mutants operated independently of Arf6 activity level. These optimized GRP1-PH constructs provide a refined biosensor system for accurate and selective detection of dynamic PIP₃ signaling, expanding the toolkit for dissecting phosphoinositide-mediated pathways. Full article

Annexins (ANNs) are a family of calcium (Ca²⁺)-dependent and phospholipid-binding proteins, which are implicated in the regulation of plant growth and development as well as protection from biotic and abiotic stresses. Corydalis saxicola Bunting, an endangered benzylisoquinoline alkaloid (BIA)-rich herbaceous plant, widely used in traditional Chinese medicine, is endemic to the calciphilic karst region of China. However, whether and how ANNs are involved in the biosynthesis pathway of BIAs and/or help C. saxicola plants cope with abiotic properties, such as calcareous soils, are largely unknown. Here, nine CsANN genes were identified from C. saxicola, and they were divided into three subfamilies, namely subfamilies I, II, and IV, based on the phylogenetic tree. The CsANNs clustered into the same clade, sharing similar gene structures and conserved motifs. The nine CsANN genes were located on five chromosomes, and their expansions were mainly attributed to tandem and whole-genome duplications. The CsANN transcripts displayed organ-specific and Ca²⁺-responsive expression patterns across various tissues. In addition, transient overexpression assays showed that CsANN1 could positively regulate the accumulation of BIA compounds in C. saxicola leaves, probably by directly interacting with key BIA-biosynthetic-pathway enzymes or by interacting with BIA-biosynthetic regulatory factors, such as MYBs. This study sheds light on the profiles and functions of the CsANN gene family and paves the way for unraveling the molecular mechanism of BIA accumulation, which is regulated by Ca²⁺ through CsANNs. Full article

Phosphatidylserine (PS) is an essential phospholipid and an emerging biomarker involved in key biological processes. While annexin V (axV) is the most widely used tool for PS detection, its calcium-dependent binding and other limitations have spurred interest in alternative probes. The lactadherin C2 domain (lactC2) offers a promising alternative, addressing many of the drawbacks associated with axV. However, its broader adoption has been hindered by challenges in production and modification for convenient experimental use. Here, we demonstrate the successful in-house engineering of fully functional recombinant bovine lactC2-based fluorescent sensors and compare their key parameters to axV probes. We show that mNeonGreen–lactC2 fusion exhibits calcium-independent binding with a comparable dissociation constant for 20% PS liposomes. We also demonstrate the detrimental effects of primary amine modification on lactC2’s PS binding efficiency, suggesting the preferential use of fluorescent protein fusion or alternative approaches. Finally, we show that unlike full-length lactadherin or axV, lactC2 inhibited thrombin generation only at high concentrations (>250 nM) in coagulation assays. These findings establish recombinant lactC2 as a versatile and promising PS sensor, with potential applications in experimental settings where axV might be unsuitable Full article

Efficient evolution exists before DNA, else the DNA genome itself could not evolve. Current data suggest RNA-membranes for this role. Selected RNAs bind well to phospholipid bilayers; randomized sequences do not. No repeated sequences are evident in selected binding RNAs. This implies small and varied membrane-affinity motifs. Such binding sequences are partially defined. Phospholipid-bound RNAs require divalents like Mg²⁺ and/or Ca²⁺, preferring more ordered bilayers: gel, ripple, or rafted membranes, in that order. RNAs also bind and stabilize bent or sharply deformed bilayers. RNA binding without divalents extends to negatively charged membranes formed from simpler anionic phospholipids and to plausibly prebiotic fatty acid bilayers. RNA-membranes frequently retain RNA solution functions: base pairing, passive transport of tryptophan, specific affinity for arginine side chains, and ribozymic ligase catalysis. Membrane-bound RNAs with several biochemical functions, linked by specific base-pairing, are readily constructed. Given these data, genetic roles seem feasible. RNA activities often require few nucleotides, easily joined in a small RNA. Base-paired groups of such RNAs can also be purposeful, joining related functions. Complex functions can therefore require only replication of short RNAs. RNA-membranes potentially segregate accurately during cell division and quickly evolve through new base pairings. Accordingly, ancient RNA-membranes could act as a protogenome, supporting encoded RNA expression, inheritance, and evolution before the DNA genome: for example, supporting organized biochemistry, coded translation, and a Standard Genetic Code. Full article

Bioenergetic membranes of mitochondria, thylakoids, and chromatophores are primary sites of ATP production in living cells. These membranes contain an electron transport chain (ETC) in which electrons are shuttled between a series of redox proteins during the generation of ATP via oxidative phosphorylation. The phospholipid composition of these membranes, which often include negative lipids, plays a role in determining the electrostatics of their surface owing to the spatial distribution of their charged head groups. Cardiolipin (CDL) is a phospholipid commonly associated with bioenergetic membranes and is also a significant contributor to the negative surface charge. Interactions between cytochromes and phospholipid head groups in the membrane can in principle affect the rate of its travel between ETC components, hence influencing the rate of ATP turnover. Here, we use molecular dynamic (MD) simulations that feature an accelerated membrane model, termed highly mobile membrane mimetic (HMMM), to study protein–lipid interactions during the diffusion of cytochrome c₂ between redox partners in a bioenergetic membrane. We observe a “skipping” mode of diffusion for cytochromes along with a bias for binding to anionic lipids, particularly with a strong preference for CDL. During diffusion, cytochrome c₂ maintains a relatively fixed tilt with respect to the membrane normal with wider fluctuations in its angle with respect to the plane of the membrane. The obtained results describing the behavior of cytochrome c₂ on a representative bioenergetic membrane have direct ramifications in shuttling motions of other similar electron-carrying elements in other bioenergetic membranes, which are composed of a significant amount of anionic lipids. The mode of surface-restricted diffusion reported here would modulate rapid electron transfer between the ETC complexes anchored in bioenergetic membranes by reducing the search space between them. Full article

Liver injury poses major health risks in livestock, necessitating effective therapeutic interventions. This study elucidates the hepatoprotective mechanisms of Euphorbia humifusa Willd. ex Schltdl. (EHW) by integrating network pharmacology, molecular docking, and experimental validation. Using a CCl₄-induced liver injury model mimicking veterinary clinical scenarios, EHW markedly alleviated hepatic damage, demonstrated by reduced liver index, serum ALT and AST levels, histopathological lesions, iron accumulation, inflammatory cytokines, and ferroptosis-associated gene expression. Network pharmacology identified EHW’s core bioactive components (quercetin, kaempferol, and β-sitosterol) and critical targets (IL-6, STAT3, HIF-1α, PTGS2, NFE2L2, and KEAP1) which were linked to ferroptosis and oxidative stress. Molecular docking revealed robust binding affinities between these compounds and ferroptosis-related proteins. In vivo validation confirmed that EHW inhibited KEAP1, activated NFE2L2-mediated antioxidant defenses (upregulating SOD1 and NQO1), restored iron homeostasis (lowering TFR1, elevating FTH1), and attenuated phospholipid peroxidation by suppressing ACSL4 and ALOX12. These results indicate that EHW mitigates ferroptosis-driven liver injury via KEAP1-NFE2L2 signaling to restore iron homeostasis and reduce oxidative stress, offering a mechanistic foundation for its clinical application in veterinary hepatoprotection. Full article

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are known to help resolve inflammation through generation of anti-inflammatory eicosanoids and specialized pro-resolving mediators, including resolvins, protectins, and maresins. Through binding to the GPR120/FFAR4 receptor, their beneficial effects result from phospholipid membrane remodeling, impairment of inflammatory signaling molecules clustering, subsequent inhibition of NF-κB and inflammasome activation, and a reduction in oxidative stress. Obesity, a chronic inflammatory disease that contributes to metabolic disorders, is alleviated by n-3 PUFAs. In the adipose tissue (AT) of individuals with obesity, n-3 PUFAs counteract hypoxia, inhibit immune cell infiltration and AT inflammation, improve insulin sensitivity, and reduce fat mass. Beyond AT, n-3 PUFAs also alleviate other metabolic disorders such as metabolic-associated steatotic liver disease (MASLD), gut dysbiosis, and/or renal dysfunction. In cardiovascular disease (CVD), they are mainly recommended as a secondary prevention for patients with coronary heart disease risks. This review provides an in-depth analysis of the benefits of n-3 PUFAs in obesity and related metabolic diseases, examining both the mechanistic and clinical aspects. Additionally, it also explores the effects of n-3 PUFAs in obesity-related chronic inflammatory conditions, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, osteoarthritis, and multiple sclerosis, by targeting specific pathophysiological mechanisms. Clinical applications and limitations of n-3 PUFAs are discussed based on findings from human clinical trials. Full article

Fermented walnut (FW) meal exhibits antifungal activity against Penicillium victoriae (the fungus responsible for prickly pear spoilage), which is mainly attributed to the synergistic effect of antimicrobial peptides and salicylic acid (SA). This study aimed to investigate the synergistic mechanism between YVVPW (YW-5, the peptide with the highest antifungal activity) and SA against the cell membrane of P. victoriae. Treatment enhanced prickly pear’s rot rate, polyphenol concentration, and superoxide dismutase (SOD) activity by 38.11%, 8.11%, and 48.53%, respectively, while reducing the microbial count by 19.17%. Structural analyses revealed β-sheets as YW-5′s predominant structure (41.18%), which increased to 49.0% during SA interaction. Molecular docking demonstrated YW-5′s stronger binding to β-(1,3)-glucan synthase and membrane protein amino acids via hydrogen bonds, hydrophobic forces, and π-π conjugate interactions. Spectroscopic analyses demonstrated SA’s major role in YW-5 synergy at the interface and polar head region of phospholipids, enhancing lipid chain disorder and the leakage of cell components. Malondialdehyde and SOD levels increased nearly two-fold and six-fold when treated with YW-5/SA, and YW-5 showed a more pronounced effect. Scanning electron and transmission electron microscopy confirmed that SA caused greater damage to spore morphology and cell ultrastructure. These findings support this formulation’s functions as an efficient antifungal substance in fruit storage. Full article

Biomimetic chromatography is a powerful tool used in the pharmaceutical industry to characterize the physicochemical properties of molecules during early drug discovery. Some studies have indicated that biomimetic chromatography may also be useful for the evaluation of toxicologically relevant molecules. In this study, we evaluated the usefulness of the biomimetic chromatography approach for determining the lipophilicity, affinity to phospholipids, and bind to plasma proteins of selected organophosphate pesticides. Quantitative structure–retention relationship (QSRR) models were proposed to understand the structural features that influence the experimentally determined properties. ACD/labs, Chemicalize, and alvaDesc software were used to calculate theoretical descriptors. Multilinear regression was used as the regression type, and feature selection was supported by a genetic algorithm. The obtained QSRR models were validated internally and externally, and they demonstrated satisfactory performance with key statistical parameters ranged from 0.844 to 0.914 for R² and 0.696–0.898 for R²_ext, respectively, indicating good predictive ability. Full article

Objectives: Annexin-A1 is a 37 kDa phospholipid-binding protein which is concentrated in a truncated 34 kDa form (AnnA1) in caveolae on the tumor vascular endothelial cell surface with expression in many tumor types. PRISM developed the monoclonal mouse antibody mAnnA1 against AnnA1 for evaluation of AnnA1 as a potential target for imaging and therapy in oncology. mAnnA1 was humanized to make hAnnA1 for translation to clinical studies. Both PRISM-produced mAnnA1 and cGMP contractor-produced hAnnA1 were investigated using noninvasive PET/CT imaging, and dosimetry was evaluated to enable clinical translation of this strategy and to investigate in vivo behavior of hAnnA1. Methods: Antibodies mAnnA1 and hAnnA1 (PRISM “hAnnA1-P” or contractor generated “hAnnA1-C”) were conjugated with the chelator deferoxamine and evaluated for immunoreactivity with ELISA. Conjugated antibodies were radiolabeled with zirconium-89. Naïve mice, rats, and non-human primates (NHP) were injected with [⁸⁹Zr]mAnnA1 or [⁸⁹Zr]hAnnA1 and imaged with PET/CT up to 10 days post injection. After imaging, mice and rats were euthanized and organs were collected, weighed, and radioactivity was quantified using a gamma counter. Dosimetry in mice and NHPs were calculated using OLINDA. Results: [⁸⁹Zr]mAnnA1 showed similar biodistribution to other antibodies with slow clearance through the liver. Transition to [⁸⁹Zr]hAnnA1-C during the dosimetry studies revealed substantial uptake in the spleen (130 ± 48% ID/g at day 5 post injection in female BALB/c), which was not observed with [⁸⁹Zr]mAnnA1 (5.6 ± 1.7% ID/g at day 7 PI). Further studies in multiple strains of mice showed variable elevated splenic uptake of [⁸⁹Zr]hAnnA1-C across mouse strains, with the highest uptake observed in female BALB/c mice (118.4 ± 23.1% ID/g) and the lowest uptake observed in male CD1 mice (34.7 ± 10.2% ID/g). Additionally, splenic uptake of hAnnA1-C was observed in Fischer rats (2.8 ± 0.6% ID/organ) and NHPs (1.6 ± 0.6% ID/organ), although at lower levels than what was observed in BALB/c mice (8.8 ± 1.8% ID/organ). Dosimetry results showed similar values between estimates based on mouse and NHP data, with the largest difference seen in the spleen (5.2 vs. 2.6 mSv/MBq in females respectively). Sequencing of hAnnA1-C revealed a frameshift mutation in the antibody sequence introduced during cGMP manufacture. Restoration of the antibody sequence by PRISM returned the antibody distribution into alignment with mAnnA1. Conclusions: An aberration introduced during cGMP production of hAnnA1-C resulted in increased splenic uptake and alteration of the biodistribution in mice. PET imaging enabled quantitative detection of the immunogenic behavior of hAnnA1, which led to detection of the sequence error. Restoration of the sequence resulted in an antibody which was non-immunogenic to mice. Full article

Background/Objectives: The novel pyrazoloquinolinone ligand CW-02-79 shows a unique profile of selective binding to σ2 receptors, but its poor solubility in both water and lipids makes its research and development a burdensome task. We aimed to develop a phospholipid-complex-based nanoemulsion formulation containing CW-02-79 suitable for intravenous administration in preclinical research. Methods: The decorated and undecorated nanoemulsions were formulated and subjected to detailed physiochemical characterization. The delivery and exposure to CW-02-79 from selected nanoemulsions were examined in the in vitro blood–brain barrier model based on human-induced pluripotent stem-cell-derived microvascular endothelial cells, astrocytes, and pericytes, and in vivo neuropharmacokinetic study in rats, respectively. Results: The developed biocompatible nanoemulsions loaded with a CW-02-79—phospholipid complex at a mass ratio of 1:10 exhibited a small droplet size and narrow size distribution, with satisfactory physicochemical stability during steam sterilization and short-term storage at 25 °C. The analysis of protein binding interactions revealed that the PEGylated nanoemulsions had fewer observable interactions compared to the undecorated nanoemulsions, especially when 0.2% DSPE-PEG2000 and 0.1% DSPE-PEG2000-mannose were combined. An in vitro BBB study demonstrated that a substantial part of CW-02-79 present in the applied nanoemulsion is able to permeate the barrier. The quantification of CW-02-79 in plasma/brain homogenate and calculated pharmacokinetic parameters confirmed good systemic and brain availability after intravenous administration. There were subtle differences in the pharmacokinetic parameters in favor of a dual surface-functionalized nanoemulson containing the glucose transporter-1-targeting ligand (mannose). Conclusions: The developed and characterized nanoemulsions enable substantial brain exposure to CW-02-79 as a prerequisite for a pharmacologically and clinically relevant selective modulation of σ2 receptors. Full article

Accidents caused by Loxosceles spiders, commonly known as brown spiders, are frequent in warm and temperate regions worldwide, with a higher prevalence in South America and the southern United States. In the venoms of species clinically associated with accidents, phospholipases D (PLDs) are the most expressed toxins. This classification is based on the toxins’ ability to cleave various phospholipids, with a preference for sphingomyelin. Studies using purified PLDs have demonstrated that these enzymes cleave phospholipids from cells, producing derivatives that can activate leukocytes. A dysregulated inflammatory response is the primary effect following envenomation, leading to dermonecrosis, which is histopathologically characterized by aseptic coagulative necrosis—a key feature of envenomation. Although advances in understanding the structure–function relationship of enzymes have been achieved through molecular biology, heterologous expression, site-directed mutations, crystallography, and bioinformatic analyses—describing PLDs in the venoms of various species and highlighting the conservation of amino acid residues involved in catalysis, substrate binding, and magnesium stabilization—little is known about the cellular biology of these PLDs. Studies have shown that the treatment of various cells with recombinant PLDs stimulates the formation of ectosomes and ectocytosis, events that initiate a cascade of intracellular signaling in PLD-binding cells and lead to the release of extracellular microvesicles. These microvesicles may act as signalosomes for other target cells, thereby triggering an inflammatory response and dermonecrosis. In this review, we will discuss the biochemical properties of PLDs, the target cells that bind to them, and the ectocytosis-dependent pathophysiology of envenoming. Full article

Cytochrome c is one of the most prominent representatives of peripheral membrane proteins. Besides functioning as an electron transfer carrier in the mitochondrial respiratory chain, it can acquire peroxidase capability, promote the self-assembly of α-synuclein, and function as a scavenger of superoxide. An understanding of its function requires knowledge of how the protein interacts with the inner membrane of mitochondria. The first part of this article provides an overview of a variety of experiments that were aimed at exploring the details of cytochrome c binding to anionic lipid liposomes, which serve as a model system for the inner membrane. While cytochrome c binding involves a conformational change from a folded into a partially disordered state, α-synuclein is intrinsically disordered in solution and subjected to a partial coil -> helix transition on membranes. Depending on the solution conditions and the surface density of α-synuclein, the protein facilitates the self-assembly into oligomers and fibrils. As for cytochrome c, results of binding experiments are discussed. In addition, the article analyzes experiments that explored α-synuclein aggregation. Similarities and differences between cytochrome c and α-synuclein binding are highlighted. Finally, the article presents a brief account of the interplay between cytochrome c and α-synuclein and its biological relevance. Full article