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Rheumatology (Oxford, England) Feb 2024Antibodies against phospholipid (aPL)-binding proteins, in particular, beta 2 glycoprotein I (β2GPI), are diagnostic/classification and pathogenic antibodies in...
Antibodies against phospholipid (aPL)-binding proteins, in particular, beta 2 glycoprotein I (β2GPI), are diagnostic/classification and pathogenic antibodies in antiphospholipid syndrome (APS). β2GPI-aPL recognize their target on endothelium and trigger a pro-thrombotic phenotype which is amplified by circulating monocytes, platelets and neutrophils. Complement activation is required as supported by the lack of aPL-mediated effects in animal models when the complement cascade is blocked. The final result is a localized clot. A strong generalized inflammatory response is associated with catastrophic APS, the clinical variant characterized by systemic thrombotic microangiopathy. A two-hit hypothesis was suggested to explain why persistent aPL are associated with acute events only when a second hit allows antibody/complement binding by modulating β2GPI tissue presentation. β2GPI/β2GPI-aPL are also responsible for obstetric APS, being the molecule physiologically present in placental/decidual tissues. Additional mechanisms mediated by aPL with different characteristics have been reported, but their diagnostic/prognostic value is still a matter of research.
Topics: Animals; Female; Pregnancy; Antiphospholipid Syndrome; Antibodies, Antiphospholipid; Placenta; Autoantibodies; Complement Activation; Thrombosis; beta 2-Glycoprotein I
PubMed: 38320591
DOI: 10.1093/rheumatology/kead603 -
BioRxiv : the Preprint Server For... Sep 2023Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized by the Kennedy pathway from choline and...
Phosphatidylcholine and phosphatidylethanolamine, the two most abundant phospholipids in mammalian cells, are synthesized by the Kennedy pathway from choline and ethanolamine, respectively. Despite the importance of these lipids, the mechanisms that enable the cellular uptake of choline and ethanolamine remain unknown. Here, we show that FLVCR1, whose mutation leads to the neurodegenerative syndrome PCARP, transports extracellular choline and ethanolamine into cells for phosphorylation by downstream kinases to initiate the Kennedy pathway. Structures of FLVCR1 in the presence of choline and ethanolamine reveal that both metabolites bind to a common binding site comprised of aromatic and polar residues. Despite binding to a common site, the larger quaternary amine of choline interacts differently with FLVCR1 than does the primary amine of ethanolamine. Structure-guided mutagenesis identified residues that are critical for the transport of ethanolamine, while being dispensable for choline transport, enabling functional separation of the entry points into the two branches of the Kennedy pathway. Altogether, these studies reveal how FLCVR1 is a high-affinity metabolite transporter that serves as the common origin for phospholipid biosynthesis by two branches of the Kennedy pathway.
PubMed: 37808796
DOI: 10.1101/2023.09.28.560019 -
PLoS Biology Dec 2023Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator involved in various physiological and pathological processes especially those related to the...
Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator involved in various physiological and pathological processes especially those related to the immune system. GPR34, GPR174, and P2Y10 have been identified as the receptors for LysoPS, and its analogues have been developed as agonists or antagonists for these receptors. However, the lack of structural information hinders the drug development with novel characteristics, such as nonlipid ligands and allosteric modulators. Here, we determined the structures of human GPR34 and GPR174 in complex with LysoPS and G protein by cryo-EM. Combined with structural analysis and functional studies, we elucidated the lipid-binding modes of these receptors. By structural comparison, we identified the structural features of GPR34 and GPR174 in active state. Taken together, our findings provide insights into ligand recognition and signaling of LysoPS receptors and will facilitate the development of novel therapeutics for related inflammatory diseases and autoimmune diseases.
Topics: Humans; Ligands; Lysophospholipids; Receptors, G-Protein-Coupled; Receptors, Lysophospholipid
PubMed: 38048360
DOI: 10.1371/journal.pbio.3002387 -
Annals of Medicine Dec 2023The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation...
AIM
The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid β1-40 (Aβ), transthyretin (TTR) variant S52P, human β2microglobulin (β2m) variants ΔN6 and D76N, Serum Amyloid A (SAA).
METHODS
The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy.
RESULTS
The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aβ, β2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aβ, β2 D76N and TTR.SPR assays show that liposomes bind Aβ and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of β2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the β-stranded peptide regions responsible for the amyloid formation.
CONCLUSION
Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases.Key messageAmyloidosis is a group of diseases, each due to a specific protein misfolding.Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders.Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.
Topics: Humans; Liposomes; Amyloid; Protein Aggregates; Molecular Chaperones; Phosphatidic Acids; Apolipoproteins
PubMed: 37143345
DOI: 10.1080/07853890.2023.2205659 -
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 -
FASEB Journal : Official Publication of... Aug 2023Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell...
Phosphatidylserine (PS) is a negatively charged phospholipid normally localized to the inner leaflet of the plasma membrane of cells but is externalized onto the cell surface during apoptosis as well as in malignant and infected cells. Consequently, PS may comprise an important molecular target in diagnostics, imaging, and targeted delivery of therapeutic agents. While an array of PS-binding molecules exist, their utility has been limited by their inability to internalize diagnostic or therapeutic payloads. We describe the generation, isolation, characterization, and utility of a PS-binding motif comprised of a carboxylated glutamic acid (GLA) residue domain that both recognizes and binds cell surface-exposed PS, and then unlike other PS-binding molecules is internalized into these cells. Internalization is independent of the traditional endosomal-lysosomal pathway, directly entering the cytosol of the target cell rapidly. We demonstrate that this PS recognition extends to stem cells and that GLA-domain-conjugated probes can be detected upon intravenous administration in animal models of infectious disease and cancer. GLA domain binding and internalization offer new opportunities for specifically targeting cells with surface-exposed PS for imaging and delivery of therapeutics.
Topics: Animals; Phosphatidylserines; Cell Membrane; Phospholipids; Phagocytosis; Neoplasms
PubMed: 37486772
DOI: 10.1096/fj.202201250RRR -
Journal For Immunotherapy of Cancer Dec 2023CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various...
BACKGROUND
CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach.
METHODS
To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography.
RESULTS
Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling.
CONCLUSION
Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.
Topics: Humans; Hepatitis A Virus Cellular Receptor 2; Single-Domain Antibodies; Phosphatidylserines; Annexin A5; T-Lymphocyte Subsets
PubMed: 38040419
DOI: 10.1136/jitc-2023-007631 -
Cell Cycle (Georgetown, Tex.) Sep 2023Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER)...
Desipramine induces eryptosis in human erythrocytes, an effect blunted by nitric oxide donor sodium nitroprusside and N-acetyl-L-cysteine but enhanced by Calcium depletion.
Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET. To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms. Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH] from high performance liquid chromatography. Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC. Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced.
Topics: Humans; Nitric Oxide Donors; Nitroprusside; Eryptosis; Calcium; Acetylcysteine; Desipramine; Erythrocytes; Glutathione; Annexins; Phosphatidylserines; Cell Size; Ceramides; Reactive Oxygen Species; Oxidative Stress
PubMed: 37522842
DOI: 10.1080/15384101.2023.2234177 -
Nature Feb 2024The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane β-barrel...
The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane β-barrel proteins (OMPs) and lipoproteins. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic β-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate β-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.
Topics: Bacterial Outer Membrane Proteins; Cell Membrane; Glycine; Lipopolysaccharides; Lipoproteins; Phospholipids; Binding Sites; Proteostasis; Lipid Bilayers; Proteome; Regulon; Protein Domains; Antimicrobial Peptides; Gram-Negative Bacteria
PubMed: 38081298
DOI: 10.1038/s41586-023-06925-5 -
Frontiers in Cellular and Infection... 2023The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by...
The African swine fever virus (ASFV) is strongly dependent on an intact endocytic pathway and a certain cellular membrane remodeling for infection, possibly regulated by the endosomal sorting complexes required for transport (ESCRT). The ESCRT machinery is mainly involved in the coordination of membrane dynamics; hence, several viruses exploit this complex and its accessory proteins VPS4 and ALIX for their own benefit. In this work, we found that shRNA-mediated knockdown of VPS4A decreased ASFV replication and viral titers, and this silencing resulted in an enhanced expression of ESCRT-0 component HRS. ASFV infection slightly increased HRS expression but not under VPS4A depletion conditions. Interestingly, VPS4A silencing did not have an impact on ALIX expression, which was significantly overexpressed upon ASFV infection. Further analysis revealed that ALIX silencing impaired ASFV infection at late stages of the viral cycle, including replication and viral production. In addition to ESCRT, the accessory protein ALIX is involved in endosomal membrane dynamics in a lysobisphosphatydic acid (LBPA) and Ca-dependent manner, which is relevant for intraluminal vesicle (ILV) biogenesis and endosomal homeostasis. Moreover, LBPA interacts with NPC2 and/or ALIX to regulate cellular cholesterol traffic, and would affect ASFV infection. Thus, we show that LBPA blocking impacted ASFV infection at both early and late infection, suggesting a function for this unconventional phospholipid in the ASFV viral cycle. Here, we found for the first time that silencing of VPS4A and ALIX affects the infection later on, and blocking LBPA function reduces ASFV infectivity at early and later stages of the viral cycle, while ALIX was overexpressed upon infection. These data suggested the relevance of ESCRT-related proteins in ASFV infection.
Topics: Swine; Animals; Endosomal Sorting Complexes Required for Transport; African Swine Fever Virus; Calcium-Binding Proteins; Endosomes; Endocytosis
PubMed: 38125905
DOI: 10.3389/fcimb.2023.1163569