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Nature Communications Aug 2023The dual functions of TMEM16F as Ca-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of...
The dual functions of TMEM16F as Ca-activated ion channel and lipid scramblase raise intriguing questions regarding their molecular basis. Intrigued by the ability of the FDA-approved drug niclosamide to inhibit TMEM16F-dependent syncytia formation induced by SARS-CoV-2, we examined cryo-EM structures of TMEM16F with or without bound niclosamide or 1PBC, a known blocker of TMEM16A Ca-activated Cl channel. Here, we report evidence for a lipid scrambling pathway along a groove harboring a lipid trail outside the ion permeation pore. This groove contains the binding pocket for niclosamide and 1PBC. Mutations of two residues in this groove specifically affect lipid scrambling. Whereas mutations of some residues in the binding pocket of niclosamide and 1PBC reduce their inhibition of TMEM16F-mediated Ca influx and PS exposure, other mutations preferentially affect the ability of niclosamide and/or 1PBC to inhibit TMEM16F-mediated PS exposure, providing further support for separate pathways for ion permeation and lipid scrambling.
Topics: Humans; Anoctamins; Calcium; Calcium Channels; Niclosamide; COVID-19; SARS-CoV-2; Lipids; Phospholipid Transfer Proteins
PubMed: 37573365
DOI: 10.1038/s41467-023-40410-x -
Nature Communications Nov 2023Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known...
Asymmetric distribution of phospholipids in eukaryotic membranes is essential for cell integrity, signaling pathways, and vesicular trafficking. P4-ATPases, also known as flippases, participate in creating and maintaining this asymmetry through active transport of phospholipids from the exoplasmic to the cytosolic leaflet. Here, we present a total of nine cryo-electron microscopy structures of the human flippase ATP8B1-CDC50A complex at 2.4 to 3.1 Å overall resolution, along with functional and computational studies, addressing the autophosphorylation steps from ATP, substrate recognition and occlusion, as well as a phosphoinositide binding site. We find that the P4-ATPase transport site is occupied by water upon phosphorylation from ATP. Additionally, we identify two different autoinhibited states, a closed and an outward-open conformation. Furthermore, we identify and characterize the PI(3,4,5)P binding site of ATP8B1 in an electropositive pocket between transmembrane segments 5, 7, 8, and 10. Our study also highlights the structural basis of a broad lipid specificity of ATP8B1 and adds phosphatidylinositol as a transport substrate for ATP8B1. We report a critical role of the sn-2 ester bond of glycerophospholipids in substrate recognition by ATP8B1 through conserved S403. These findings provide fundamental insights into ATP8B1 catalytic cycle and regulation, and substrate recognition in P4-ATPases.
Topics: Humans; Adenosine Triphosphatases; Substrate Specificity; Cryoelectron Microscopy; Phospholipid Transfer Proteins; Phospholipids; Adenosine Triphosphate; Cell Membrane
PubMed: 37980352
DOI: 10.1038/s41467-023-42828-9 -
The Journal of Cell Biology Dec 2023Sphingomyelin plays a key role in cellular cholesterol homeostasis by binding to and sequestering cholesterol in the plasma membrane. We discovered that synthesis of...
Sphingomyelin plays a key role in cellular cholesterol homeostasis by binding to and sequestering cholesterol in the plasma membrane. We discovered that synthesis of very long chain (VLC) sphingomyelins is inversely regulated by cellular cholesterol levels; acute cholesterol depletion elicited a rapid induction of VLC-sphingolipid synthesis, increased trafficking to the Golgi apparatus and plasma membrane, while cholesterol loading reduced VLC-sphingolipid synthesis. This sphingolipid-cholesterol metabolic axis is distinct from the sterol responsive element binding protein pathway as it requires ceramide synthase 2 (CerS2) activity, epidermal growth factor receptor signaling, and was unaffected by inhibition of protein translation. Depletion of VLC-ceramides reduced plasma membrane cholesterol content, reduced plasma membrane lipid packing, and unexpectedly resulted in the accumulation of cholesterol in the cytoplasmic leaflet of the lysosome membrane. This study establishes the existence of a cholesterol-sphingolipid regulatory axis that maintains plasma membrane lipid homeostasis via regulation of sphingomyelin synthesis and trafficking.
Topics: Cell Membrane; Cytoplasm; Homeostasis; Intracellular Membranes; Sphingomyelins; Sphingosine N-Acyltransferase; Cholesterol; ErbB Receptors
PubMed: 37787764
DOI: 10.1083/jcb.202308055 -
The EMBO Journal Sep 2023Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby...
Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby exposing to the cytosol intraluminal, otherwise shielded danger signals such as glycans and sphingomyelin. The detection of glycans by galectin-8 triggers anti-bacterial autophagy, but how cells sense and respond to cytosolically exposed sphingomyelin remains unknown. Here, we identify TECPR1 (tectonin beta-propeller repeat containing 1) as a receptor for cytosolically exposed sphingomyelin, which recruits ATG5 into an E3 ligase complex that mediates lipid conjugation of LC3 independently of ATG16L1. TECPR1 binds sphingomyelin through its N-terminal DysF domain (N'DysF), a feature not shared by other mammalian DysF domains. Solving the crystal structure of N'DysF, we identified key residues required for the interaction, including a solvent-exposed tryptophan (W154) essential for binding to sphingomyelin-positive membranes and the conjugation of LC3 to lipids. Specificity of the ATG5/ATG12-E3 ligase responsible for the conjugation of LC3 is therefore conferred by interchangeable receptor subunits, that is, the canonical ATG16L1 and the sphingomyelin-specific TECPR1, in an arrangement reminiscent of certain multi-subunit ubiquitin E3 ligases.
Topics: Animals; Sphingomyelins; Microtubule-Associated Proteins; Autophagy-Related Proteins; Carrier Proteins; Autophagy; Ubiquitin-Protein Ligases; Autophagy-Related Protein 5; Mammals
PubMed: 37409490
DOI: 10.15252/embj.2022113012 -
The Journal of Cell Biology Oct 2023Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is...
Mitochondria are dynamic organelles regulated by fission and fusion processes. The fusion of membranes requires elaborative coordination of proteins and lipids and is particularly crucial for the function and quality control of mitochondria. Phosphatidic acid (PA) on the mitochondrial outer membrane generated by PLD6 facilitates the fusion of mitochondria. However, how PA promotes mitochondrial fusion remains unclear. Here, we show that a mitochondrial outer membrane protein, NME3, is required for PLD6-induced mitochondrial tethering or clustering. NME3 is enriched at the contact interface of two closely positioned mitochondria depending on PLD6, and NME3 binds directly to PA-exposed lipid packing defects via its N-terminal amphipathic helix. The PA binding function and hexamerization confer NME3 mitochondrial tethering activity. Importantly, nutrient starvation enhances the enrichment efficiency of NME3 at the mitochondrial contact interface, and the tethering ability of NME3 contributes to fusion efficiency. Together, our findings demonstrate NME3 as a tethering protein promoting selective fusion between PLD6-remodeled mitochondria for quality control.
Topics: Humans; Mitochondria; Mitochondrial Dynamics; Mitochondrial Membranes; Mitochondrial Proteins; NM23 Nucleoside Diphosphate Kinases; Phosphatidic Acids; Phospholipase D
PubMed: 37584589
DOI: 10.1083/jcb.202301091 -
Biomolecules Aug 2023Oxidative stress is a well-known hallmark of Antiphospholipid Antibody Syndrome (APS), a systemic autoimmune disease characterized by arterial and venous thrombosis... (Review)
Review
Oxidative stress is a well-known hallmark of Antiphospholipid Antibody Syndrome (APS), a systemic autoimmune disease characterized by arterial and venous thrombosis and/or pregnancy morbidity. Oxidative stress may affect various signaling pathways and biological processes, promoting dysfunctional immune responses and inflammation, inducing apoptosis, deregulating autophagy and impairing mitochondrial function. The chronic oxidative stress and the dysregulation of the immune system leads to the loss of tolerance, which drives autoantibody production and inflammation with the development of endothelial dysfunction. In particular, anti-phospholipid antibodies (aPL), which target phospholipids and/or phospholipid binding proteins, mainly β-glycoprotein I (β-GPI), play a functional role in the cell signal transduction pathway(s), thus contributing to oxidative stress and thrombotic events. An oxidation-antioxidant imbalance may be detected in the blood of patients with APS as a reflection of disease progression. This review focuses on functional evidence highlighting the role of oxidative stress in the initiation and progression of APS. The protective role of food supplements and Nuclear Factor Erythroid 2-Related Factor 2 (NRF2) activators in APS patients will be summarized to point out the potential of these therapeutic approaches to reduce APS-related clinical complications.
Topics: Female; Pregnancy; Humans; Antibodies, Antiphospholipid; NF-E2-Related Factor 2; Oxidative Stress; Phospholipids; Antioxidants
PubMed: 37627286
DOI: 10.3390/biom13081221 -
Nature Communications Oct 2023Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients....
Cryptococcus spp. are environmental fungi that first must adapt to the host environment before they can cause life-threatening meningitis in immunocompromised patients. Host CO concentrations are 100-fold higher than the external environment and strains unable to grow at host CO concentrations are not pathogenic. Using a genetic screening and transcriptional profiling approach, we report that the TOR pathway is critical for C. neoformans adaptation to host CO partly through Ypk1-dependent remodeling of phosphatidylserine asymmetry at the plasma membrane. We also describe a C. neoformans ABC/PDR transporter (PDR9) that is highly expressed in CO-sensitive environmental strains, suppresses CO-induced phosphatidylserine/phospholipid remodeling, and increases susceptibility to host concentrations of CO. Interestingly, regulation of plasma membrane lipid asymmetry by the TOR-Ypk1 axis is distinct in C. neoformans compared to S. cerevisiae. Finally, host CO concentrations suppress the C. neoformans pathways that respond to host temperature (Mpk1) and pH (Rim101), indicating that host adaptation requires a stringent balance among distinct stress responses.
Topics: Humans; Cryptococcus neoformans; Saccharomyces cerevisiae; Phospholipids; Carbon Dioxide; Phosphatidylserines; Cryptococcosis; ATP-Binding Cassette Transporters
PubMed: 37852972
DOI: 10.1038/s41467-023-42318-y -
Nature Communications Sep 2023The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is...
The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is activated by caspase-mediated cleavage and binding of the XRCC4 fragment. Here, we show that extracellular calcium is an additional factor needed to activate Xkr4. The constitutively active mutant of Xkr4 is found to induce phospholipid scrambling in an extracellular, but not intracellular, calcium-dependent manner. Importantly, other Xkr family members also require extracellular calcium for activation. Alanine scanning shows that D123 and D127 of TM1 and E310 of TM3 coordinate calcium binding. Moreover, lysine scanning demonstrates that the E310K mutation-mediated salt bridge between TM1 and TM3 bypasses the requirement of calcium. Cysteine scanning proves that disulfide bond formation between TM1 and TM3 also activates phospholipid scrambling without calcium. Collectively, this study shows that extracellular calcium functions as a molecular glue for TM1 and TM3 of Xkr proteins for activation, thus demonstrating a regulatory mechanism for multi-transmembrane region-containing proteins.
Topics: Calcium; Alanine; Biological Transport; Caspases; Phosphatidylserines
PubMed: 37696806
DOI: 10.1038/s41467-023-40934-2 -
Nature Communications Apr 2024Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent...
Oxidative stress-induced lipid accumulation is mediated by lipid droplets (LDs) homeostasis, which sequester vulnerable unsaturated triglycerides into LDs to prevent further peroxidation. Here we identify the upregulation of lipopolysaccharide-binding protein (LBP) and its trafficking through LDs as a mechanism for modulating LD homeostasis in response to oxidative stress. Our results suggest that LBP induces lipid accumulation by controlling lipid-redox homeostasis through its lipid-capture activity, sorting unsaturated triglycerides into LDs. N-acetyl-L-cysteine treatment reduces LBP-mediated triglycerides accumulation by phospholipid/triglycerides competition and Peroxiredoxin 4, a redox state sensor of LBP that regulates the shuttle of LBP from LDs. Furthermore, chronic stress upregulates LBP expression, leading to insulin resistance and obesity. Our findings contribute to the understanding of the role of LBP in regulating LD homeostasis and against cellular peroxidative injury. These insights could inform the development of redox-based therapies for alleviating oxidative stress-induced metabolic dysfunction.
Topics: Acute-Phase Proteins; Carrier Proteins; Homeostasis; Lipid Droplets; Lipopolysaccharides; Membrane Glycoproteins; Oxidative Stress; Triglycerides
PubMed: 38615060
DOI: 10.1038/s41467-024-47553-5 -
The Journal of Biological Chemistry Dec 2023Vacuolar H-ATPases (V-ATPases) are highly conserved multisubunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is...
Vacuolar H-ATPases (V-ATPases) are highly conserved multisubunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue- and organelle-specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle-specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms, and we hypothesize that the aNT domains of these isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. We determined that bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP-binding sites, we identified potential binding sites in the HuaNT domains by sequence comparisons and existing subunit structures and models. We found that mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles.
Topics: Humans; Binding Sites; Endosomes; Golgi Apparatus; Hydrogen-Ion Concentration; Lysosomes; Phosphatidylinositol Phosphates; Protein Isoforms; Protein Subunits; Saccharomyces cerevisiae; Substrate Specificity; Vacuolar Proton-Translocating ATPases; Protein Domains
PubMed: 37979916
DOI: 10.1016/j.jbc.2023.105473