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Nanoscale May 2024Lipid coating is considered a versatile strategy to equip nanoparticles (NPs) with a biomimetic surface coating, but the membrane properties of these nanoassemblies...
Lipid coating is considered a versatile strategy to equip nanoparticles (NPs) with a biomimetic surface coating, but the membrane properties of these nanoassemblies remain in many cases insufficiently understood. In this work, we apply C-Laurdan generalized polarization (GP) measurements to probe the temperature-dependent polarity of hybrid membranes consisting of a lipid monolayer adsorbed onto a polylactic acid (PLA) polymer core as function of lipid composition and compare the behavior of the lipid coated NPs (LNPs) with that of liposomes assembled from identical lipid mixtures. The LNPs were generated by nanoprecipitation of the polymer in aqueous solutions containing two types of lipid mixtures: (i) cholesterol, dipalmitoylphosphatidylcholine (DPPC), and the ganglioside GM3, as well as (ii) dioleoylphosphatidylcholine (DOPC), DPPC and GM3. LNPs were found to exhibit more distinct and narrower phase transitions than corresponding liposomes and to retain detectable phase transitions even for cholesterol or DOPC concentrations that yielded no detectable transitions in liposomes. These findings together with higher GP values in the case of the LNPs for temperatures above the phase transition temperature indicate a stabilization of the membrane through the polymer core. LNP binding studies to GM3-recognizing cells indicate that differences in the membrane fluidity affect binding avidity in the investigated model system.
Topics: Polyesters; Nanoparticles; Membrane Fluidity; Liposomes; Cholesterol; Polymers; 1,2-Dipalmitoylphosphatidylcholine; Lactic Acid; Lipids; Temperature; G(M3) Ganglioside
PubMed: 38595322
DOI: 10.1039/d3nr06464f -
MBio May 2024Systemic infections by spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug...
UNLABELLED
Systemic infections by spp. are associated with high mortality rates, partly due to limitations in current antifungals, highlighting the need for novel drugs and drug targets. The fungal phosphatidylserine synthase, Cho1, from is a logical antifungal drug target due to its importance in virulence, absence in the host, and conservation among fungal pathogens. Inhibitors of Cho1 could serve as lead compounds for drug development, so we developed a target-based screen for inhibitors of purified Cho1. This enzyme condenses serine and cytidyldiphosphate-diacylglycerol (CDP-DAG) into phosphatidylserine (PS) and releases cytidylmonophosphate (CMP). Accordingly, we developed an nucleotidase-coupled malachite-green-based high throughput assay for purified Cho1 that monitors CMP production as a proxy for PS synthesis. Over 7,300 molecules curated from repurposing chemical libraries were interrogated in primary and dose-responsivity assays using this platform. The screen had a promising average ' score of ~0.8, and seven compounds were identified that inhibit Cho1. Three of these, ebselen, LOC14, and CBR-5884, exhibited antifungal effects against cells, with fungicidal inhibition by ebselen and fungistatic inhibition by LOC14 and CBR-5884. Only CBR-5884 showed evidence of disrupting Cho1 function by inducing phenotypes consistent with the mutant, including a reduction of cellular PS levels. Kinetics curves and computational docking indicate that CBR-5884 competes with serine for binding to Cho1 with a of 1,550 ± 245.6 nM. Thus, this compound has the potential for development into an antifungal compound.
IMPORTANCE
Fungal phosphatidylserine synthase (Cho1) is a logical antifungal target due to its crucial role in the virulence and viability of various fungal pathogens, and since it is absent in humans, drugs targeted at Cho1 are less likely to cause toxicity in patients. Using fungal Cho1 as a model, there have been two unsuccessful attempts to discover inhibitors for Cho1 homologs in whole-cell screens prior to this study. The compounds identified in these attempts do not act directly on the protein, resulting in the absence of known Cho1 inhibitors. The significance of our research is that we developed a high-throughput target-based assay and identified the first Cho1 inhibitor, CBR-5884, which acts both on the purified protein and its function in the cell. This molecule acts as a competitive inhibitor with a value of 1,550 ± 245.6 nM and, thus, has the potential for development into a new class of antifungals targeting PS synthase.
Topics: Candida albicans; Antifungal Agents; CDPdiacylglycerol-Serine O-Phosphatidyltransferase; Enzyme Inhibitors; High-Throughput Screening Assays; Small Molecule Libraries; Microbial Sensitivity Tests; Fungal Proteins; Phosphatidylserines; Furans; Thiophenes
PubMed: 38587428
DOI: 10.1128/mbio.00633-24 -
Nutrition Research and Practice Apr 2024High levels of plasma low-density lipoprotein (LDL) cholesterol are an important determinant of atherosclerotic lesion formation. The disruption of cholesterol efflux or...
BACKGROUND/OBJECTIVES
High levels of plasma low-density lipoprotein (LDL) cholesterol are an important determinant of atherosclerotic lesion formation. The disruption of cholesterol efflux or reverse cholesterol transport (RCT) in peripheral tissues and macrophages may promote atherogenesis. The aim of the current study was to examine whether bioactive ellagic acid, a functional food component, improved RCT functionality and high-density lipoprotein (HDL) function in diet-induced atherogenesis of apolipoproteins E (apoE) knockout (KO) mice.
MATERIALS/METHODS
Wild type mice and apoE KO mice were fed a high-cholesterol Paigen diet for 10 weeks to induce hypercholesterolemia and atherosclerosis, and concomitantly received 10 mg/kg ellagic acid via gavage.
RESULTS
Supplying ellagic acid enhanced induction of apoE and ATP-binding cassette (ABC) transporter G1 in oxidized LDL-exposed macrophages, facilitating cholesterol efflux associated with RCT. Oral administration of ellagic acid to apoE KO mice fed on Paigen diet improved hypercholesterolemia with reduced atherogenic index. This compound enhanced the expression of ABC transporters in peritoneal macrophages isolated from apoE KO mice fed on Paigen diet, indicating increased cholesterol efflux. Plasma levels of cholesterol ester transport protein and phospholipid transport protein involved in RCT were elevated in mice lack of apoE gene, which was substantially reduced by supplementing ellagic acid to Paigen diet-fed mice. In addition, ellagic acid attenuated hepatic lipid accumulation in apoE KO mice, evidenced by staining of hematoxylin and eosin and oil red O. Furthermore, the supplementation of 10 mg/kg ellagic acid favorably influenced the transcriptional levels of hepatic LDL receptor and scavenger receptor-B1 in Paigen diet-fed apoE KO mice.
CONCLUSION
Ellagic acid may be an athero-protective dietary compound encumbering diet-induced atherogenesis though improving the RCT functionality.
PubMed: 38584811
DOI: 10.4162/nrp.2024.18.2.194 -
Molecular Therapy : the Journal of the... Jun 2024Second mitochondrial-derived activator of caspase (SMAC), also known as direct inhibitor of apoptosis-binding proteins with low pI (Diablo), is known as a pro-apoptotic...
Second mitochondrial-derived activator of caspase (SMAC), also known as direct inhibitor of apoptosis-binding proteins with low pI (Diablo), is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions, including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell-penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in vivo study, a survivin/baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5)-derived peptide was selected, due to its overexpression in many cancers and its involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD-8 cells and increased cell-intrinsic programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents an innovative therapeutic cancer paradigm.
Topics: Humans; Survivin; Animals; Mice; Mitochondrial Proteins; Apoptosis; Cell Line, Tumor; Cell Proliferation; Apoptosis Regulatory Proteins; Intracellular Signaling Peptides and Proteins; Neoplasms; Inflammation; Xenograft Model Antitumor Assays; Protein Binding; Inhibitor of Apoptosis Proteins; Cell-Penetrating Peptides; Peptides; Immunosuppression Therapy
PubMed: 38582961
DOI: 10.1016/j.ymthe.2024.04.007 -
Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.Cell Reports Apr 2024CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages,...
CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24 ovarian cancers.
Topics: Animals; Female; Humans; Mice; Acyltransferases; Amidohydrolases; CD24 Antigen; Cell Line, Tumor; Glycosylphosphatidylinositols; Macrophages; Ovarian Neoplasms; Phagocytosis
PubMed: 38573857
DOI: 10.1016/j.celrep.2024.114041 -
ACS Chemical Biology Apr 2024Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous...
Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca with a ∼ 45 μM. Each subsequent binding affinity ( ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca binding affinity, we explored the extent that Ca binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) and dioleoylphosphatidylserine (DOPS) positively modulated Ca binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) was reduced with increasing [Ca]. Overall, we find that specific lipids differentially modulate Ca binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca, and anionic phospholipids that may also control some aspects of vesicular exocytosis.
Topics: Calcium; Exocytosis; Neurotransmitter Agents; Phospholipids; Synaptic Transmission; Synaptic Vesicles; Synaptotagmin I; Animals; Rats
PubMed: 38566504
DOI: 10.1021/acschembio.3c00772 -
The EMBO Journal May 2024The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain...
The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P and PI(3,4,5)P attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.
Topics: Humans; Breast Neoplasms; Transcription Factors; YAP-Signaling Proteins; Female; Trans-Activators; Adaptor Proteins, Signal Transducing; Signal Transduction; Phosphoproteins; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Cell Line, Tumor; Phosphatidylinositol Phosphates; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Gene Expression Regulation, Neoplastic; DNA-Binding Proteins; Cell Nucleus; Phosphotransferases (Alcohol Group Acceptor); Intracellular Signaling Peptides and Proteins
PubMed: 38565949
DOI: 10.1038/s44318-024-00085-6 -
ACS Physical Chemistry Au Mar 2024Intrinsically disordered regions of proteins are responsible for many biological processes such as in the case of liver kinase B1 (LKB1)-a serine/threonine kinase...
Intrinsically disordered regions of proteins are responsible for many biological processes such as in the case of liver kinase B1 (LKB1)-a serine/threonine kinase relevant for cell proliferation and cell polarity. LKB1 becomes fully activated upon recruitment to the plasma membrane by binding of its disordered C-terminal polybasic motif consisting of eight lysines/arginines to phospholipids. Here, we present extensive molecular dynamics (MD) simulations of the polybasic motif interacting with a model membrane composed of 1-palmitoyl-2-oleoyl--glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl phosphatidic acid (PA) and cell culture experiments. Protein-membrane binding effects are due to the electrostatic interactions between the polybasic amino acids and PAs. For significant binding, the first three lysines turn out to be dispensable, which was also recapitulated in cell culture using transfected GFP-LKB1 variants. LKB1-membrane binding results in nonmonotonous changes in the structure of the protein as well as the membrane, in particular, accumulation of PAs and reduced thickness at the protein-membrane contact area. The protein-lipid binding turns out to be highly dynamic due to an interplay of PA-PA repulsion and protein-PA attraction. The thermodynamics of this interplay is captured by a statistical fluctuation model, which allows the estimation of both energies. Quantification of the significance of each polar amino acid in the polybasic provides detailed insights into the molecular mechanism of protein-membrane binding of LKB1. These results can likely be transferred to other proteins, which interact by intrinsically disordered polybasic regions with anionic membranes.
PubMed: 38560754
DOI: 10.1021/acsphyschemau.3c00051 -
Journal of Obesity 2024Obesity is expected to hinder efferocytosis due to ADAM17-mediated cleavage of the MER tyrosine kinase receptor, producing soluble MER (sMER) that disrupts MERTK binding...
BACKGROUND
Obesity is expected to hinder efferocytosis due to ADAM17-mediated cleavage of the MER tyrosine kinase receptor, producing soluble MER (sMER) that disrupts MERTK binding to cell death markers. However, the intracellular efferocytosis pathway in central obesity remains elusive, particularly the role of low-grade chronic inflammation in its initiation and identification of binding signals that disrupt efferocytosis.
OBJECTIVE
We investigate the efferocytosis signaling pathway in men with central obesity and its relationship with inflammation, cell death, and related processes.
METHODS
A cross-sectional study was conducted, and clinical data and blood samples were collected from 56 men with central obesity (obese group) and 29 nonobese individuals (control group). Clinical evaluations and predefined biochemical screening tests were performed. The efferocytosis signaling pathway was investigated by measuring phosphatidylserine (PS), ADAM17, TNF-alpha (TNF-), and sMER.
RESULTS
Metabolic syndrome was detected in more than half of the participants in the obese group according to the predefined tests. Mean levels of PS, TNF-, and sMER were higher in the obese group but not significantly different from those of the control group. Further analysis based on waist circumference (WC) ranges in the obese group revealed a significant increase in PS and sMER levels between the control group and the obese group with WC greater than 120 cm. ADAM17 levels were significantly higher in the obese group than in the control group. PS was positively correlated with WC and ADAM17. ADAM17 was positively correlated with TNF- and sMER, indicating impaired efferocytosis.
CONCLUSIONS
Central obesity appeared to cause a disturbance in efferocytosis that began with cell damage and death, along with an enlargement of the WC and an ongoing inflammatory response. Efferocytosis was disrupted by proinflammatory cytokine regulators, which induced the production of sMER and interfered with the efferocytosis process.
Topics: Humans; Male; ADAM17 Protein; Cross-Sectional Studies; Efferocytosis; Inflammation; Obesity, Abdominal; Phagocytosis; Phosphatidylserines; Tumor Necrosis Factor-alpha
PubMed: 38550672
DOI: 10.1155/2024/1424404 -
Life (Basel, Switzerland) Feb 2024The PI3K enzymes modify phospholipids to regulate cell growth and differentiation. Somatic variants in PI3K are recurrent in cancer and drive a proliferative phenotype....
The PI3K enzymes modify phospholipids to regulate cell growth and differentiation. Somatic variants in PI3K are recurrent in cancer and drive a proliferative phenotype. Somatic mosaicism of and are associated with vascular anomalies and overgrowth syndromes. Germline variants are associated with varying phenotypes, including immunodeficiency or facial dysmorphism with growth delay, lipoatrophy, and insulin resistance associated with SHORT syndrome. There has been limited study of the molecular mechanism to unify our understanding of how variants in drive both undergrowth and overgrowth phenotypes. Thus, we compiled genomic variants from cancer and rare vascular anomalies and sought to interpret their effects using an unbiased physics-based simulation approach for the protein complex. We applied molecular dynamics simulations to mechanistically understand how genetic variants affect PIK3R1 and its interactions with PIK3CA. Notably, iSH2 genetic variants associated with undergrowth destabilize molecular interactions with the PIK3CA receptor binding domain in simulations, which is expected to decrease activity. On the other hand, overgrowth and cancer variants lead to loss of inhibitory interactions in simulations, which is expected to increase activity. We find that all disease variants display dysfunctions on either structural characteristics or intermolecular interaction energy. Thus, this comprehensive characterization of novel mosaic somatic variants associated with two opposing phenotypes has mechanistic importance and biomedical relevance and may aid in future therapeutic developments.
PubMed: 38541623
DOI: 10.3390/life14030297