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PLoS Pathogens Jun 2024COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly...
COVID-associated coagulopathy seemly plays a key role in post-acute sequelae of SARS- CoV-2 infection. However, the underlying pathophysiological mechanisms are poorly understood, largely due to the lack of suitable animal models that recapitulate key clinical and pathological symptoms. Here, we fully characterized AC70 line of human ACE2 transgenic (AC70 hACE2 Tg) mice for SARS-CoV-2 infection. We noted that this model is highly permissive to SARS-CoV-2 with values of 50% lethal dose and infectious dose as ~ 3 and ~ 0.5 TCID50 of SARS-CoV-2, respectively. Mice infected with 105 TCID50 of SARS-CoV-2 rapidly succumbed to infection with 100% mortality within 5 days. Lung and brain were the prime tissues harboring high viral titers, accompanied by histopathology. However, viral RNA and inflammatory mediators could be detectable in other organs, suggesting the nature of a systemic infection. Lethal challenge of AC70 hACE2 Tg mice caused acute onset of leukopenia, lymphopenia, along with an increased neutrophil-to-lymphocyte ratio (NLR). Importantly, infected animals recapitulated key features of COVID-19-associated coagulopathy. SARS-CoV-2 could induce the release of circulating neutrophil extracellular traps (NETs), along with activated platelet/endothelium marker. Immunohistochemical staining with anti-platelet factor-4 (PF4) antibody revealed profound platelet aggregates especially within blocked veins of the lungs. We showed that acute SARS-CoV-2 infection triggered a hypercoagulable state coexisting with ill-regulated fibrinolysis. Finally, we highlighted the potential role of Annexin A2 (ANXA2) in fibrinolytic failure. ANXA2 is a calcium-dependent phospholipid-binding protein that forms a heterotertrameric complexes localized at the extracellular membranes with two S100A10 small molecules acting as a co-receptor for tissue-plasminogen activator (t-PA), tightly involved in cell surface fibrinolysis. Thus, our results revealing elevated IgG type anti-ANXA2 antibody production, downregulated de novo ANXA2/S100A10 synthesis, and reduced ANXA2/S100A10 association in infected mice, this protein might serve as druggable targets for development of antithrombotic and/or anti-fibrinolytic agents to attenuate pathogenesis of COVID-19.
PubMed: 38913740
DOI: 10.1371/journal.ppat.1011777 -
Biophysical Journal Jun 2024The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug...
The ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) is a multidrug efflux pump that is overexpressed in a variety of cancers and associated with the drug resistance phenomenon. P-gp structures were previously determined in detergent and in nanodiscs, in which different transmembrane helix conformations were found, "straight" and "kinked", respectively, indicating a possible role of the lipid environment on the P-gp structural ensemble. Here, we investigate the dynamic conformational ensembles and protein-lipid interactions of two human P-gp inward-open conformers, straight and kinked, employing all-atom molecular dynamics simulations in asymmetric multicomponent lipid bilayers that mimic the highly specialized hepatocyte membrane in which P-gp is expressed. The two conformers are found to differ in terms of the accessibility of the substrate cavity. The MD simulations show how cholesterol and different lipid species wedge, snorkel, and partially enter within the cavity of the straight P-gp conformer solved in detergent. However, the access to the cavity of kinked P-gp conformer solved in nanodiscs is restricted. Furthermore, the volume and dynamic fluctuations of the substrate cavity largely differ between the two P-gp structures, and are modulated by the presence (or absence) of cholesterol in the membrane and/or of ATP. From the mechanistic perspective, the findings indicate that the straight conformer likely precedes the kinked conformer in the functional working cycle of P-gp, with the latter conformation representing a post substrate-bound state. The inaccessibility of the main transmembrane cavity in the kinked conformer might be crucial in preventing substrate disengagement and transport withdrawal. Remarkably, in our unbiased MD simulations, one transmembrane helix (TM10) of the straight conformer underwent a spontaneous conformational transition to a kinked conformation, underlining the relevance of both conformations in a native phospholipid environment and revealing structural descriptors defining the transition between two P-gp conformers.
PubMed: 38909280
DOI: 10.1016/j.bpj.2024.06.020 -
Nature Communications Jun 2024Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which...
Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solve the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discover that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane lipid packing defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function.
Topics: Endosomes; Cryoelectron Microscopy; Phosphatidylinositol Phosphates; Membrane Fusion; rab5 GTP-Binding Proteins; Humans; Vesicular Transport Proteins; Cell Membrane; Animals; Lysosomes
PubMed: 38898033
DOI: 10.1038/s41467-024-49137-9 -
Nature Communications Jun 2024Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require...
Autophagy is relevant for diverse processes in eukaryotic cells, making its regulation of fundamental importance. The formation and maturation of autophagosomes require a complex choreography of numerous factors. The endosomal sorting complex required for transport (ESCRT) is implicated in the final step of autophagosomal maturation by sealing of the phagophore membrane. ESCRT-III components were shown to mediate membrane scission by forming filaments that interact with cellular membranes. However, the molecular mechanisms underlying the recruitment of ESCRTs to non-endosomal membranes remain largely unknown. Here we focus on the ESCRT-associated protein ALG2-interacting protein X (ALIX) and identify Ca-dependent lipid binding protein 1 (CaLB1) as its interactor. Our findings demonstrate that CaLB1 interacts with AUTOPHAGY8 (ATG8) and PI(3)P, a phospholipid found in autophagosomal membranes. Moreover, CaLB1 and ALIX localize with ATG8 on autophagosomes upon salt treatment and assemble together into condensates. The depletion of CaLB1 impacts the maturation of salt-induced autophagosomes and leads to reduced delivery of autophagosomes to the vacuole. Here, we propose a crucial role of CaLB1 in augmenting phase separation of ALIX, facilitating the recruitment of ESCRT-III to the site of phagophore closure thereby ensuring efficient maturation of autophagosomes.
Topics: Arabidopsis; Autophagosomes; Endosomal Sorting Complexes Required for Transport; Arabidopsis Proteins; Calcium-Binding Proteins; Autophagy; Phosphatidylinositol Phosphates; Autophagy-Related Protein 8 Family; Vacuoles; Phase Separation
PubMed: 38898014
DOI: 10.1038/s41467-024-49485-6 -
Journal of Clinical Medicine May 2024Antiphospholipid syndrome (APS), also known as Hughes syndrome, is an acquired autoimmune and procoagulant condition that predisposes individuals to recurrent thrombotic... (Review)
Review
Antiphospholipid syndrome (APS), also known as Hughes syndrome, is an acquired autoimmune and procoagulant condition that predisposes individuals to recurrent thrombotic events and obstetric complications. Central is the role of three types of antiphospholipid antibodies that target phospholipid-binding proteins: lupus anticoagulant (LAC), anti-β2-glycoprotein I (β2-GPI-Ab), and anti-cardiolipin (aCL). Together with clinical data, these antibodies are the diagnostic standard. However, the diagnosis of APS in older adults may be challenging and, in the diagnostic workup of thromboembolic complications, it is an underestimated etiology. The therapeutic management of APS requires distinguishing two groups with differential risks of thromboembolic complications. The standard therapy is based on low-dose aspirin in the low-risk group and vitamin K antagonists in the high-risk group. The value of direct oral anticoagulants is currently controversial. The potential role of monoclonal antibodies is investigated. For example, rituximab is currently recommended in catastrophic antiphospholipid antibody syndrome. Research is ongoing on other monoclonal antibodies, such as daratumumab and obinutuzumab. This narrative review illustrates the pathophysiological mechanisms of APS, with a particular emphasis on cardiovascular complications and their impact in older adults. This article also highlights advancements in the diagnosis, risk stratification, and management of APS.
PubMed: 38892776
DOI: 10.3390/jcm13113064 -
Plants (Basel, Switzerland) May 2024In plant models such as , phosphatidic acid (PA), a key molecule of lipid signaling, was shown not only to be involved in stress responses, but also in plant development...
In plant models such as , phosphatidic acid (PA), a key molecule of lipid signaling, was shown not only to be involved in stress responses, but also in plant development and nutrition. In this article, we highlight lipid signaling existing in crop species. Based on open access databases, we update the list of sequences encoding phospholipases D, phosphoinositide-dependent phospholipases C, and diacylglycerol-kinases, enzymes that lead to the production of PA. We show that structural features of these enzymes from model plants are conserved in equivalent proteins from selected crop species. We then present an in-depth discussion of the structural characteristics of these proteins before focusing on PA binding proteins. For the purpose of this article, we consider RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), the most documented PA target proteins. Finally, we present pioneering experiments that show, by different approaches such as monitoring of gene expression, use of pharmacological agents, ectopic over-expression of genes, and the creation of silenced mutants, that lipid signaling plays major roles in crop species. Finally, we present major open questions that require attention since we have only a perception of the peak of the iceberg when it comes to the exciting field of phospholipid signaling in plants.
PubMed: 38891340
DOI: 10.3390/plants13111532 -
Nature Communications Jun 2024The transient receptor potential canonical type 3 (TRPC3) channel plays a pivotal role in regulating neuronal excitability in the brain via its constitutive activity....
The transient receptor potential canonical type 3 (TRPC3) channel plays a pivotal role in regulating neuronal excitability in the brain via its constitutive activity. The channel is intricately regulated by lipids and has previously been demonstrated to be positively modulated by PIP. Using molecular dynamics simulations and patch clamp techniques, we reveal that PIP predominantly interacts with TRPC3 at the L3 lipid binding site, located at the intersection of pre-S1 and S1 helices. We demonstrate that PIP sensing involves a multistep mechanism that propagates from L3 to the pore domain via a salt bridge between the TRP helix and S4-S5 linker. Notably, we find that both stimulated and constitutive TRPC3 activity require PIP. These structural insights into the function of TRPC3 are invaluable for understanding the role of the TRPC subfamily in health and disease, in particular for cardiovascular diseases, in which TRPC3 channels play a major role.
Topics: TRPC Cation Channels; Humans; Molecular Dynamics Simulation; Phosphatidylinositol 4,5-Diphosphate; HEK293 Cells; Binding Sites; Animals; Patch-Clamp Techniques; Protein Binding
PubMed: 38890374
DOI: 10.1038/s41467-024-49396-6 -
Nature Communications Jun 2024Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well...
Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.
Topics: Talin; Focal Adhesions; Cell Membrane; Vinculin; Humans; Animals; Phosphatidylinositol 4,5-Diphosphate; Integrins; Cytoplasm; Protein Binding; Phase Separation
PubMed: 38862544
DOI: 10.1038/s41467-024-49222-z -
BMC Genomics Jun 2024Phospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in...
BACKGROUND
Phospholipases constitute a diverse category of enzymes responsible for the breakdown of phospholipids. Their involvement in signal transduction with a pivotal role in plant development and stress responses is well documented.
RESULTS
In the present investigation, a thorough genome-wide analysis revealed that the pearl millet genome contains at least 44 phospholipase genes distributed across its 7 chromosomes, with chromosome one harbouring the highest number of these genes. The synteny analysis suggested a close genetic relationship of pearl millet phospholipases with that of foxtail millet and sorghum. All identified genes were examined to unravel their gene structures, protein attributes, cis-regulatory elements, and expression patterns in two pearl millet genotypes contrasting for rancidity. All the phospholipases have a high alpha-helix content and distorted regions within the predicted secondary structures. Moreover, many of these enzymes possess binding sites for both metal and non-metal ligands. Additionally, the putative promoter regions associated with these genes exhibit multiple copies of cis-elements specifically responsive to biotic and abiotic stress factors and signaling molecules. The transcriptional profiling of 44 phospholipase genes in two genotypes contrasting for rancidity across six key tissues during pearl millet growth revealed a predominant expression in grains, followed by seed coat and endosperm. Specifically, the genes PgPLD-alpha1-1, PgPLD-alpha1-5, PgPLD-delta1-7a, PgPLA1-II-1a, and PgPLD-delta1-2a exhibited notable expression in grains of both the genotypes while showing negligible expression in the other five tissues. The sequence alignment of putative promoters revealed several variations including SNPs and InDels. These variations resulted in modifications to the corresponding cis-acting elements, forming distinct transcription factor binding sites suggesting the transcriptional-level regulation for these five genes in pearl millet.
CONCLUSIONS
The current study utilized a genome-wide computational analysis to characterize the phospholipase gene family in pearl millet. A comprehensive expression profile of 44 phospholipases led to the identification of five grain-specific candidates. This underscores a potential role for at least these five genes in grain quality traits including the regulation of rancidity in pearl millet. Therefore, this study marks the first exploration highlighting the possible impact of phospholipases towards enhancing agronomic traits in pearl millet.
Topics: Pennisetum; Phospholipases; Multigene Family; Edible Grain; Gene Expression Regulation, Plant; Promoter Regions, Genetic; Phylogeny; Plant Proteins; Synteny; Gene Expression Profiling; Genotype; Chromosome Mapping
PubMed: 38858648
DOI: 10.1186/s12864-024-10504-x -
BioRxiv : the Preprint Server For... Jun 2024The nuclear receptor Liver Receptor Homolog-1 (LRH-1, ) binds to phospholipids that regulate important LRH-1 functions in the liver. A recent compound screen...
The nuclear receptor Liver Receptor Homolog-1 (LRH-1, ) binds to phospholipids that regulate important LRH-1 functions in the liver. A recent compound screen unexpectedly identified bilirubin, the product of liver heme metabolism, as a possible ligand for LRH-1. Here, we show unconjugated bilirubin directly binds LRH-1 with apparent =9.3uM, altering LRH-1 interaction with all transcriptional coregulator peptides tested. Bilirubin decreased LRH-1 protease sensitivity, consistent with MD simulations predicting bilirubin stably binds LRH-1 within the canonical ligand binding site. Bilirubin activated a luciferase reporter specific for LRH-1, dependent on co-expression with the bilirubin membrane transporter , but bilirubin failed to activate ligand-binding genetic mutants of LRH-1. Gene profiling in HepG2 cells shows bilirubin selectively regulated transcripts from endogenous LRH-1 ChIP-seq target genes, which was significantly attenuated by either genetic knockdown of LRH-1, or by a specific chemical competitor of LRH-1. Gene set enrichment suggests bilirubin and LRH-1 share roles in cholesterol metabolism and lipid efflux, thus we propose a new role for LRH-1 in directly sensing intracellular levels of bilirubin.
PubMed: 38853895
DOI: 10.1101/2024.05.05.592606