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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 -
Colloids and Surfaces. B, Biointerfaces Jun 2024The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or...
The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or phosphatidylserine (PS) aggregates. In PS-based assemblies, the dyes demonstrate a concentration-dependent fluorescent response, distinguishing anionic proteins such as bovine serum albumin (BSA) and pepsin from lysozyme (LYZ) in aqueous solutions. Conversely, no significant response is observed when the dyes are incorporated into the well-organized bilayers of neutral PC. The fluorescent response arises from the binding of dyes to proteins, leading to the detachment of dye molecules from the assemblies, rather than from the binding of proteins to the assemblies, although the latter process is facilitated by electrostatic attraction. Thus, both the poor ordering of PS molecules and the interfacial arrangement of the dyes are prerequisites for the fluorescent response of dye-PS aggregates. The structure of the dyes significantly impacts the spectral features of dye-PS and dye-protein assemblies. An optimal dye structure has been identified for the recognition of BSA, with a limit of detection (LOD) of 10.8 nM.
PubMed: 38908044
DOI: 10.1016/j.colsurfb.2024.114046 -
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 Biochemistry Jun 2024Cytidine diphosphate diacylglycerol (CDP-DAG) is a critical intermediate that is converted to multiple phospholipids in prokaryotes and eukaryotes. In budding yeast,...
Cytidine diphosphate diacylglycerol (CDP-DAG) is a critical intermediate that is converted to multiple phospholipids in prokaryotes and eukaryotes. In budding yeast, CDP-DAG synthesis from cytidine triphosphate (CTP) and phosphatidic acid (PA) is catalyzed by the membrane-integrated protein Cds1 in the endoplasmic reticulum and the peripheral membrane-bound protein Tam41 in mitochondria. Although a recent study revealed that the fission yeast SpTam41 consists of a nucleotidyltransferase domain and a winged helix domain, forming an active-site pocket for CTP binding between the two domains together with a C-terminal amphipathic helix for membrane association, how CTP and Mg2+, a most-favored divalent cation, are accommodated with PA remains obscure. A more recent report by Kimura et al. (J. Biochem. 2022; 171:429-441) solved the crystal structure of FbTam41, a functional ortholog from a Firmicutes bacterium, with CTP-Mg2+, successfully providing a detailed molecular view of CDP-DAG synthesis. In this commentary, our current understanding of Tam41-mediated reaction is discussed.
PubMed: 38896689
DOI: 10.1093/jb/mvae046 -
Protein Science : a Publication of the... Jul 2024Alzheimer's disease is the fastest-growing neurodegenerative disease that affects over six million Americans. The abnormal aggregation of amyloid β peptide and Tau...
Alzheimer's disease is the fastest-growing neurodegenerative disease that affects over six million Americans. The abnormal aggregation of amyloid β peptide and Tau protein is the expected molecular cause of the loss of neurons in brains of AD patients. A growing body of evidence indicates that lipids can alter the aggregation rate of amyloid β peptide and modify the toxicity of amyloid β aggregates. However, the role of lipids in Tau aggregation remains unclear. In this study, we utilized a set of biophysical methods to determine the extent to which phospatidylserine (PS) altered the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N terminal inserts that enhance the binding of Tau to tubulin. We found that the length and saturation of fatty acids (FAs) in PS altered the aggregation rate of 2N4R isoform, while no changes in the aggregation rate of 1N4R were observed. These results indicate that N terminal inserts play an important role in protein-lipid interactions. We also found that PS could change the toxicity of 1N4R and 2N4R Tau fibrils, as well as alter molecular mechanisms by which these aggregates exert cytotoxicity to neurons. Finally, we found that although Tau fibrils formed in the presence and absence of PS endocytosed by cells, only fibril species that were formed in the presence of PS exert strong impairment of the cell mitochondria.
Topics: tau Proteins; Humans; Phosphatidylserines; Tubulin; Alzheimer Disease; Protein Binding; Neurons; Protein Aggregates; Protein Isoforms
PubMed: 38895991
DOI: 10.1002/pro.5078 -
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