The Electrostatic Basis of Diacylglycerol Pyrophosphate—Protein Interaction

Diacylglycerol pyrophosphate (DGPP) is an anionic phospholipid formed in plants, yeast, and parasites under multiple stress stimuli. It is synthesized by the phosphorylation action of phosphatidic acid (PA) kinase on phosphatidic acid, a signaling lipid with multifunctional properties. PA functions in the membrane through the interaction of its negatively charged phosphomonoester headgroup with positively charged proteins and ions. DGPP, like PA, can interact electrostatically via the electrostatic-hydrogen bond switch mechanism but differs from PA in its overall charge and shape. The formation of DGPP from PA alters the physicochemical properties as well as the structural dynamics of the membrane. This potentially impacts the molecular and ionic binding of cationic proteins and ions with the DGPP enriched membrane. However, the results of these important interactions in the stress response and in DGPP’s overall intracellular function is unknown. Here, using 31P MAS NMR, we analyze the effect of the interaction of low DGPP concentrations in model membranes with the peptides KALP23 and WALP23, which are flanked by positively charged Lysine and neutral Tryptophan residues, respectively. Our results show a significant effect of KALP23 on the charge of DGPP as compared to WALP23. There was, however, no significant effect on the charge of the phosphomonoester of DGPP due to the interaction with positively charged lipids, dioleoyl trimethylammonium propane (DOTAP) and dioleoyl ethyl-phosphatidylcholine (EtPC). Divalent calcium and magnesium cations induce deprotonation of the DGPP headgroup but showed no noticeable differences on DGPP’s charge. Our results lead to a novel model for DGPP—protein interaction.

Conformational dynamics of free and membrane-bound human Hsp70 in model cytosolic and endo-lysosomal environments

The binding of the major stress-inducible human 70-kDa heat shock protein (Hsp70) to the anionic phospholipid bis-(monoacylglycero)-phosphate (BMP) in the lysosomal membrane is crucial for its impact on cellular pathology in lysosomal storage disorders. However, the conformational features of this protein-lipid complex remain unclear. Here, we apply hydrogen–deuterium exchange mass spectrometry (HDX-MS) to describe the dynamics of the full-length Hsp70 in the cytosol and its conformational changes upon translocation into lysosomes. Using wild-type and W90F mutant proteins, we also map and discriminate the interaction of Hsp70 with BMP and other lipid components of the lysosomal membrane. We identify the N-terminal of the nucleotide binding domain (residues 87–118) as the primary orchestrator of BMP interaction. We show that the conformation of this domain is significantly reorganized in the W90F mutant, explaining its inability to stabilize lysosomal membranes. Overall, our results reveal important new molecular details of the protective effect of Hsp70 in lysosomal storage diseases, which, in turn, could guide future drug development.

Pathophysiology of the Antiphospholipid Antibody Syndrome

The antiphospholipid syndrome is characterized by antibodies directed against phospholipid-binding proteins and phospholipids attached to cell membrane receptors, mitochondria, oxidized lipoproteins, and activated complement components. When antibodies bind to these complex antigens, cells are activated and the coagulation and complement cascades are triggered, culminating in thrombotic events and pregnancy morbidity that further define the syndrome. The phospholipid-binding proteins most often involved are annexins II and V, β2-glycoprotein I, prothrombin, and cardiolipin. A distinguishing feature of the antiphospholipid syndrome is the “lupus anticoagulant”. This is not a single entity but rather a family of antibodies directed against complex antigens consisting of β2-glycoprotein I and/or prothrombin bound to an anionic phospholipid. Although these antibodies prolong in vitro clotting times by competing with clotting factors for phospholipid binding sites, they are not associated with clinical bleeding. Rather, they are thrombogenic because they augment thrombin production in vivo by concentrating prothrombin on phospholipid surfaces. Other antiphospholipid antibodies decrease the clot-inhibitory properties of the endothelium and enhance platelet adherence and aggregation. Some are atherogenic because they increase lipid peroxidation by reducing paraoxonase activity, and others impair fetal nutrition by diminishing placental antithrombotic and fibrinolytic activity. This plethora of destructive autoantibodies is currently managed with immunomodulatory agents, but new approaches to treatment might include vaccines against specific autoantigens, blocking the antibodies generated by exposure to cytoplasmic DNA, and selective targeting of aberrant B-cells to reduce or eliminate autoantibody production.

Anticoagulation Targeting Membrane-Bound Anionic Phospholipids Improved Outcomes of Traumatic Brain Injury in Mice

Severe traumatic brain injury (TBI) often causes an acute systemic hypercoagulable state that rapidly develops into consumptive coagulopathy. We have recently demonstrated that TBI-induced coagulopathy (TBI-IC) is initiated and disseminated by brain-derived extracellular vesicles (BDEVs) and propagated by extracellular vesicles (EVs) from endothelial cells and platelets. Here, we present results from a study designed to test the hypothesis that anticoagulation targeting anionic phospholipid-expressing EVs prevents TBI-IC and improves the outcomes of mice subjected to severe TBI. We evaluated the effects of a fusion protein (ANV-6L15) for improving the outcomes of TBI. ANV-6L15 combines the phosphatidylserine (PS)-binding annexin V with a peptide anticoagulant modified to preferentially target extrinsic coagulation. We found that ANV-6L15 reduced intracranial hematoma by 70.2%, improved neurological function, and reduced death by 56.8% in mice subjected to fluid percussion injury at 1.9 atm. It protected the TBI mice by preventing vascular leakage, tissue edema, and the TBI-induced hypercoagulable state. We further showed that the extrinsic tenase complex was formed on the surfaces of circulating EVs, with the highest level found on BDEVs. Phospholipidomic analysis detected the highest levels of PS on BDEVs, as compared to EVs from endothelial cells and platelets (79.1, 15.2, and 3.5 nM/mg of protein, respectively). These findings demonstrate that TBI-IC results from a trauma-induced hypercoagulable state and may be treated by anticoagulation targeting on the anionic phospholipid-expressing membrane of EVs from the brain and other cells.

Inhibition of Very Long Chain Fatty Acids Synthesis Mediates PI3P Homeostasis at Endosomal Compartments

A main characteristic of sphingolipids is the presence of a very long chain fatty acid (VLCFA) whose function in cellular processes is not yet fully understood. VLCFAs of sphingolipids are involved in the intracellular traffic to the vacuole and the maturation of early endosomes into late endosomes is one of the major pathways for vacuolar traffic. Additionally, the anionic phospholipid phosphatidylinositol-3-phosphate (PtdIns (3)P or PI3P) is involved in protein sorting and recruitment of small GTPase effectors at late endosomes/multivesicular bodies (MVBs) during vacuolar trafficking. In contrast to animal cells, PI3P mainly localizes to late endosomes in plant cells and to a minor extent to a discrete sub-domain of the plant’s early endosome (EE)/trans-Golgi network (TGN) where the endosomal maturation occurs. However, the mechanisms that control the relative levels of PI3P between TGN and MVBs are unknown. Using metazachlor, an inhibitor of VLCFA synthesis, we found that VLCFAs are involved in the TGN/MVB distribution of PI3P. This effect is independent from either synthesis of PI3P by PI3-kinase or degradation of PI(3,5)P2 into PI3P by the SUPPRESSOR OF ACTIN1 (SAC1) phosphatase. Using high-resolution live cell imaging microscopy, we detected transient associations between TGNs and MVBs but VLCFAs are not involved in those interactions. Nonetheless, our results suggest that PI3P might be transferable from TGN to MVBs and that VLCFAs act in this process.

Membrane Nanoscopic Organization of D2L Dopamine Receptor Probed by Quantum Dot Tracking

The role of lateral mobility and nanodomain organization of G protein-coupled receptors in modulating subcellular signaling has been under increasing scrutiny. Investigation of D2 dopamine receptor diffusion dynamics is of particular interest, as these receptors have been linked to altered neurotransmission in affective disorders and represent the primary target for commonly prescribed antipsychotics. Here, we applied our single quantum dot tracking approach to decipher intrinsic diffusion patterns of the wild-type long isoform of the D2 dopamine receptor and its genetic variants previously identified in several cohorts of schizophrenia patients. We identified a subtle decrease in the diffusion rate of the Val96Ala mutant that parallels its previously reported reduced affinity for potent neuroleptics clozapine and chlorpromazine. Slower Val96Ala variant diffusion was not accompanied by a change in receptor-receptor transient interactions as defined by the diffraction-limited quantum dot colocalization events. In addition, we implemented a Voronoї tessellation-based algorithm to compare nanoclustering of the D2 dopamine receptor to the dominant anionic phospholipid phosphatidylinositol 4,5-bisphosphate in the plasma membrane of live cells.

Chemokines act as phosphatidylserine-bound “find-me” signals in apoptotic cell clearance

Removal of apoptotic cells is essential for maintenance of tissue homeostasis. Chemotactic cues termed “find-me” signals attract phagocytes toward apoptotic cells, which selectively expose the anionic phospholipid phosphatidylserine (PS) and other “eat-me” signals to distinguish healthy from apoptotic cells for phagocytosis. Blebs released by apoptotic cells can deliver find-me signals; however, the mechanism is poorly understood. Here, we demonstrate that apoptotic blebs generated in vivo from mouse thymus attract phagocytes using endogenous chemokines bound to the bleb surface. We show that chemokine binding to apoptotic cells is mediated by PS and that high affinity binding of PS and other anionic phospholipids is a general property of many but not all chemokines. Chemokines are positively charged proteins that also bind to anionic glycosaminoglycans (GAGs) on cell surfaces for presentation to leukocyte G protein–coupled receptors (GPCRs). We found that apoptotic cells down-regulate GAGs as they up-regulate PS on the cell surface and that PS-bound chemokines, unlike GAG-bound chemokines, are able to directly activate chemokine receptors. Thus, we conclude that PS-bound chemokines may serve as find-me signals on apoptotic vesicles acting at cognate chemokine receptors on leukocytes.

Synaptotagmin rings as high sensitivity regulators of synaptic vesicle docking and fusion

Synchronous release at neuronal synapses is accomplished by a machinery that senses calcium influx and fuses the synaptic vesicle and plasma membranes to release neurotransmitters. Previous studies suggested the calcium sensor Synaptotagmin (Syt) is a facilitator of vesicle docking and both a facilitator and inhibitor of fusion. On phospholipid monolayers, the Syt C2AB domain spontaneously oligomerized into rings that are disassembled by Ca2+, suggesting Syt rings may clamp fusion as membrane-separating "washers" until Ca2+-mediated disassembly triggers fusion and release (Wang et al., 2014). Here we combined mathematical modeling with experiment to measure mechanical properties of Syt rings and to test this mechanism. Consistent with experiment, the model quantitatively recapitulates observed Syt ring-induced dome and volcano shapes on phospholipid monolayers, and predicts rings are stabilized by anionic phospholipid bilayers or bulk solution with ATP. The selected ring conformation is highly sensitive to membrane composition and bulk ATP levels, a property that may regulate vesicle docking and fusion in ATP-rich synaptic terminals. We find the Syt molecules hosted by a synaptic vesicle oligomerize into a halo, unbound from the vesicle, but in proximity to sufficiently PIP2-rich plasma membrane (PM) domains the PM-bound trans Syt ring conformation is preferred. Thus, the Syt halo serves as landing gear for spatially directed docking at PIP2-rich sites that define the active zones of exocytotic release, positioning the Syt ring to clamp fusion and await calcium. Our results suggest the Syt ring is both a Ca2+-sensitive fusion clamp and a high-fidelity sensor for directed docking.