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Plasma membrane (PM) domains and order phases have been shown to play a key role in the assembly, release, and entry of several lipid-enveloped viruses. In the present study, we provide a mechanistic understanding of the Ebola virus (EBOV) matrix protein VP40 interaction with PM lipids and their effect on VP40 oligomerization, a crucial step for viral assembly and budding. VP40 matrix formation is sufficient to induce changes in the PM fluidity. We demonstrate that the distance between the lipid headgroups, the fatty acid tail saturation and the PM order are important factors for the stability of VP40 binding and oligomerization at the PM. Use of FDA-approved drugs to fluidize the PM, destabilizes the viral matrix assembly leading to a reduction in budding efficiency. Overall, these findings support an EBOV assembly mechanism that reaches beyond lipid headgroup specificity by using ordered PM lipid regions independent of cholesterol., Competing Interests: Conflict of interest Authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Ebola virus matrix protein VP40 is responsible for the formation of the viral matrix by localizing at the inner leaflet of the human plasma membrane (PM). Various lipid types, including PI(4,5)P 2 (i.e. PIP 2 ) and phosphatidylserine (PS), play active roles in this process. Specifically, the negatively charged headgroups of both PIP 2 and PS interact with the basic residues of VP40 and stabilize it at the membrane surface, allowing for eventual egress. Phosphatidic acid (PA), resulting from the enzyme phospholipase D (PLD), is also known to play an active role in viral development. In this work, we performed a biophysical and computational analysis to investigate the effects of the presence of PA on the membrane localization and association of VP40. We used coarse-grained molecular dynamics simulations to quantify VP40 hexamer interactions with the inner leaflet of the PM. Analysis of the local distribution of lipids shows enhanced lipid clustering when PA is abundant in the membrane. We observed that PA lipids have a similar role to that of PS lipids in VP40 association due to the geometry and charge. Complementary experiments performed in cell culture demonstrate competition between VP40 and a canonical PA-binding protein for the PM. Also, inhibition of PA synthesis reduced the detectable budding of virus-like particles. These computational and experimental results provide new insights into the early stages of Ebola virus budding and the role that PA lipids have on the VP40-PM association., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and is lethal in a large percentage of those infected. The EBOV matrix protein viral protein 40 kDa (VP40) is a peripheral binding protein that forms a shell beneath the lipid bilayer in virions and virus-like particles (VLPs). VP40 is required for virus assembly and budding from the host cell plasma membrane. VP40 is a dimer that can rearrange into oligomers at the plasma membrane interface, but it is unclear how these structures form and how they are stabilized. We therefore investigated the ability of VP40 to form stable oligomers using in vitro and cellular assays. We characterized two lysine-rich regions in the VP40 C-terminal domain (CTD) that bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) and play distinct roles in lipid binding and the assembly of the EBOV matrix layer. The extensive analysis of VP40 with and without lipids by hydrogen deuterium exchange mass spectrometry revealed that VP40 oligomers become extremely stable when VP40 binds PI(4,5)P 2 . The PI(4,5)P 2 -induced stability of VP40 dimers and oligomers is a critical factor in VP40 oligomerization and release of VLPs from the plasma membrane. The two lysine-rich regions of the VP40 CTD have different roles with respect to interactions with plasma membrane phosphatidylserine (PS) and PI(4,5)P 2 . CTD region 1 (Lys221, Lys224, and Lys225) interacts with PI(4,5)P 2 more favorably than PS and is important for VP40 extent of oligomerization. In contrast, region 2 (Lys270, Lys274, Lys275, and Lys279) mediates VP40 oligomer stability via lipid interactions and has a more prominent role in release of VLPs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of the article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A series of pyrene-benzothiazolium dyes (1a-1d) were experimentally investigated to study their internalization mechanism into cellular lysosomes as well as their potential imaging applications for live cell imaging. The lysosome selectivity of the probes was further compared by using fluorescently tagged lysosome associated membrane protein-1 (LAMP-1) expression-dependent visualization in both normal (COS-7, HEK293) and cancer (A549, Huh 7.5) cell lines. These probes were successfully employed as reliable lysosome markers in tumor cell models, thus providing an attractive alternative to LAMP-1 expression-dependent visualization methods. One advantage of these probes is the elimination of significant background fluorescence arising from fluorescently tagged protein expression on the cell surface when cells were transfected with LAMP-1 expression plasmids. Probes exhibited remarkable ability to stain cellular lysosomes for long-term experiments (up to 24 h) and the highly lipophilic nature of the probe design allowed their accumulation in hydrophobic regions of the cellular lysosomes. Experimental evidences indicated that the probes are likely to be internalized into lysosomes via endocytosis and accumulated in the hydrophobic regions of the lysosomes rather than in the acidic lysosomal lumen. These probes also demonstrated significant stability and lysosome staining for fixed cell imaging applications as well. Lastly, the benzothiazolium moiety of the probes was identified as the key component for lysosome selectivity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Artemisinin resistance threatens worldwide malaria control and elimination. Elevation of phosphatidylinositol-3-phosphate (PI3P) can induce resistance in blood stages of Plasmodium falciparum The parasite unfolded protein response (UPR) has also been implicated as a proteostatic mechanism that may diminish artemisinin-induced toxic proteopathy. How PI3P acts and its connection to the UPR remain unknown, although both are conferred by mutation in P falciparum Kelch13 (K13), the marker of artemisinin resistance. Here we used cryoimmunoelectron microscopy to show that K13 concentrates at PI3P tubules/vesicles of the parasite's endoplasmic reticulum (ER) in infected red cells. K13 colocalizes and copurifies with the major virulence adhesin PfEMP1. The PfEMP1-K13 proteome is comprehensively enriched in multiple proteostasis systems of protein export, quality control, and folding in the ER and cytoplasm and UPR. Synthetic elevation of PI3P that induces resistance in absence of K13 mutation also yields signatures of proteostasis and clinical resistance. These findings imply a key role for PI3P-vesicle amplification as a mechanism of resistance of infected red cells. As validation, the major resistance mutation K13C580Y quantitatively increased PI3P tubules/vesicles, exporting them throughout the parasite and the red cell. Chemical inhibitors and fluorescence microscopy showed that alterations in PfEMP1 export to the red cell and cytoadherence of infected cells to a host endothelial receptor are features of multiple K13 mutants. Together these data suggest that amplified PI3P vesicles disseminate widespread proteostatic capacity that may neutralize artemisinins toxic proteopathy and implicate a role for the host red cell in artemisinin resistance. The mechanistic insights generated will have an impact on malaria drug development., (© 2018 by The American Society of Hematology.)

Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic., (Copyright © 2017 Elsevier Inc. All rights reserved.)

To reduce costs of lipid-binding assays, allow for multiple lipids to be screened for protein binding simultaneously, and to make lipid binding more user friendly, lipids have been dotted onto membranes to investigate lipid-protein interactions. These assays are similar to a western blot where the membrane is blocked, incubated with a protein of interest and detected using antibodies. Although the assay is inexpensive and straightforward, problems with promiscuous or poor binding, as well as insufficient blocking occur frequently. In this technical note, we share several specific improvements to ensure lipid-protein overlay assays are of high quality and contain proper controls., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In this issue of Chemistry & Biology, Antal and colleagues describe how phosphorylation optimizes the signaling range of protein kinase C (PKC) isoforms. Priming of these enzymes regulates intramolecular conformational changes, which reduces access to their diacylglycerol (DAG) binding C1 domains., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Group IVA cytosolic phospholipase A₂ (cPLA₂α), which harbors an N-terminal lipid binding C2 domain and a C-terminal lipase domain, produces arachidonic acid from the sn-2 position of zwitterionic lipids such as phosphatidylcholine. The C2 domain has been shown to bind zwitterionic lipids, but more recently, the anionic phosphomonoester sphingolipid metabolite ceramide-1-phosphate (C1P) has emerged as a potent bioactive lipid with high affinity for a cationic patch in the C2 domain β-groove. To systematically analyze the role that C1P plays in promoting the binding of cPLA₂α-C2 to biological membranes, we employed biophysical measurements and cellular translocation studies along with mutagenesis. Biophysical and cellular translocation studies demonstrate that C1P specificity is mediated by Arg⁵⁹, Arg⁶¹, and His⁶² (an RxRH sequence) in the C2 domain. Computational studies using molecular dynamics simulations confirm the origin of C1P specificity, which results in a spatial shift of the C2 domain upon membrane docking to coordinate the small C1P headgroup. Additionally, the hydroxyl group on the sphingosine backbone plays an important role in the interaction with the C2 domain, further demonstrating the selectivity of the C2 domain for C1P over phosphatidic acid. Taken together, this is the first study demonstrating the molecular origin of C1P recognition.

Group IVA cytosolic phospholipase A(2) (cPLA(2)α) is an 85 kDa enzyme that regulates the release of arachidonic acid (AA) from the sn-2 position of membrane phospholipids. It is well established that cPLA(2)α binds zwitterionic lipids such as phosphatidylcholine in a Ca(2+)-dependent manner through its N-terminal C2 domain, which regulates its translocation to cellular membranes. In addition to its role in AA synthesis, it has been shown that cPLA(2)α promotes tubulation and vesiculation of the Golgi and regulates trafficking of endosomes. Additionally, the isolated C2 domain of cPLA(2)α is able to reconstitute Fc receptor-mediated phagocytosis, suggesting that C2 domain membrane binding is sufficient for phagosome formation. These reported activities of cPLA(2)α and its C2 domain require changes in membrane structure, but the ability of the C2 domain to promote changes in membrane shape has not been reported. Here we demonstrate that the C2 domain of cPLA(2)α is able to induce membrane curvature changes to lipid vesicles, giant unilamellar vesicles, and membrane sheets. Biophysical assays combined with mutagenesis of C2 domain residues involved in membrane penetration demonstrate that membrane insertion by the C2 domain is required for membrane deformation, suggesting that C2 domain-induced membrane structural changes may be an important step in signaling pathways mediated by cPLA(2)α.

Phosphatidylinositol 4-phosphate (PtdIns(4)P) lipid is an essential component of eukaryotic membranes and a marker of the Golgi complex. Here, we developed metabolically stabilized (ms) analogs of PtdIns(4)P and the inositol 1,4-bisphosphate (IP(2)) head group derivative and demonstrated that these compounds can substitute the natural lipid fully retaining its physiological activities. The methylenephosphonate (MP) and phosphorothioate (PT) analogs of PtdIns(4)P and the aminohexyl (AH)-IP(2) probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms analogs and IP(2) probes could be invaluable for identifying protein effectors and characterizing PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN)., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The spatial and temporal regulation of lipid molecules in cell membranes is a hallmark of cellular signaling and membrane trafficking events. Lipid-mediated targeting provides for strict control and versatility, because cell membranes harbor a large number of lipid molecules with variation in head group and acyl chain structures. Signaling and trafficking proteins contain a large number of modular domains that exhibit specific lipid binding properties and play a critical role in their localization and function. Nearly 20 years of research including structural, computational, biochemical and biophysical studies have demonstrated how these lipid-binding domains recognize their target lipid and achieve subcellular localization. The integration of this individual lipid-binding domain data in the context of the full-length proteins, macromolecular signaling complexes, and the lipidome is only beginning to be unraveled and represents a target of therapeutic development. This review brings together recent findings and classical concepts to concisely summarize the lipid-binding domain field while illustrating where the field is headed and how the gaps may be filled in with new technologies.

The general receptor for phosphoinositides isoform 1 (GRP1) is recruited to the plasma membrane in response to activation of phosphoinositide 3-kinases and accumulation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. GRP1's pleckstrin homology (PH) domain recognizes PtdIns(3,4,5)P(3) with high specificity and affinity, however, the precise mechanism of its association with membranes remains unclear. Here, we detail the molecular basis of membrane anchoring by the GRP1 PH domain. Our data reveal a multivalent membrane docking involving PtdIns(3,4,5)P(3) binding, regulated by pH and facilitated by electrostatic interactions with other anionic lipids. The specific recognition of PtdIns(3,4,5)P(3) triggers insertion of the GRP1 PH domain into membranes. An acidic environment enhances PtdIns(3,4,5)P(3) binding and increases membrane penetration as demonstrated by NMR and monolayer surface tension and surface plasmon resonance experiments. The GRP1 PH domain displays a 28 nM affinity for POPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/PtdIns(3,4,5)P(3) vesicles at pH 6.0, but binds 22-fold weaker at pH 8.0. The pH sensitivity is attributed in part to the His355 residue, protonation of which is required for the robust interaction with PtdIns(3,4,5)P(3) and significant membrane penetration, as illustrated by mutagenesis data. The binding affinity of the GRP1 PH domain for PtdIns(3,4,5)P(3)-containing vesicles is further amplified (by approximately 6-fold) by nonspecific electrostatic interactions with phosphatidylserine/phosphatidylinositol. Together, our results provide new insight into the multivalent mechanism of the membrane targeting and regulation of the GRP1 PH domain.

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2alpha). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P2 in decreasing the dissociation constant of the cPLA2alpha-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P2, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P2 activated cPLA2alpha with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P2, but not C1P, increased the penetration ability of cPLA2alpha into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA2alpha by anionic lipids. First, C1P activates cPLA2alpha by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P2 activates the enzyme by increasing catalytic efficiency through increased membrane penetration.

Epsin and AP180 are essential components of the endocytotic machinery, which controls internalization of protein receptors and other macromolecules at the cell surface. Epsin and AP180 are recruited to the plasma membrane by their structurally and functionally related N-terminal ENTH and ANTH domains that specifically recognize PtdIns(4,5)P2. Here, we show that membrane anchoring of the ENTH and ANTH domains is regulated by the acidic environment. Lowering the pH enhances PtdIns(4,5)P2 affinity of the ENTH and ANTH domains reinforcing their association with lipid vesicles and monolayers. The pH dependency is due to the conserved histidine residues of the ENTH and ANTH domains, protonation of which is necessary for the strong PtdIns(4,5)P2 recognition, as revealed by liposome binding, surface plasmon resonance, NMR, monolayer surface tension and mutagenesis experiments. The pH sensitivity of the ENTH and ANTH domains is reminiscent to the pH dependency of the FYVE domain suggesting a common regulatory mechanism of membrane anchoring by a subset of the PI-binding domains.

Ceramide kinase (CERK) is a critical mediator of eicosanoid synthesis, and its product, ceramide-1-phosphate (C1P), is required for the production of prostaglandins in response to several inflammatory agonists. In this study, mass spectrometry analysis disclosed that the main forms of C1P in cells were C(16:0) C1P and C(18:0) C1P, suggesting that CERK uses ceramide transported to the trans-Golgi apparatus by ceramide transport protein (CERT). To this end, downregulation of CERT by RNA interference technology dramatically reduced the levels of newly synthesized C1P (kinase-derived) as well as significantly reduced the total mass levels of C1P in cells. Confocal microscopy, subcellular fractionation, and surface plasmon resonance analysis were used to further localize CERK to the trans-Golgi network, placing the generation of C1P in the proper intracellular location for the recruitment of cytosolic phospholipase A(2)alpha. In conclusion, these results demonstrate that CERK localizes to areas of eicosanoid synthesis and uses a ceramide "pool" transported in an active manner via CERT.

Membrane-binding peripheral proteins play important roles in many biological processes, including cell signaling and membrane trafficking. Unlike integral membrane proteins, these proteins bind the membrane mostly in a reversible manner. Since peripheral proteins do not have canonical transmembrane segments, it is difficult to identify them from their amino acid sequences. As a first step toward genome-scale identification of membrane-binding peripheral proteins, we built a kernel-based machine learning protocol. Key features of known membrane-binding proteins, including electrostatic properties and amino acid composition, were calculated from their amino acid sequences and tertiary structures, which were then incorporated into the support vector machine to perform the classification. A data set of 40 membrane-binding proteins and 230 non-membrane-binding proteins was used to construct and validate the protocol. Cross-validation and holdout evaluation of the protocol showed that the accuracy of the prediction reached up to 93.7% and 91.6%, respectively. The protocol was applied to the prediction of membrane-binding properties of four C2 domains from novel protein kinases C. Although these C2 domains have 50% sequence identity, only one of them was predicted to bind the membrane, which was verified experimentally with surface plasmon resonance analysis. These results suggest that our protocol can be used for predicting membrane-binding properties of a wide variety of modular domains and may be further extended to genome-scale identification of membrane-binding peripheral proteins.

FYVE domains are membrane targeting domains that are found in proteins involved in endosomal trafficking and signal transduction pathways. Most FYVE domains bind specifically to phosphatidylinositol 3-phosphate (PI(3)P), a lipid that resides mainly in endosomal membranes. Though the specific interactions between FYVE domains and the headgroup of PI(3)P have been well characterized, principally through structural studies, the available experimental structures suggest several different models for FYVE/membrane association. Thus, the manner in which FYVE domains adsorb to the membrane surface remains to be elucidated. Towards this end, recent experiments have shown that FYVE domains bind PI(3)P in the context of phospholipid bilayers and that hydrophobic residues on a conserved loop are able to penetrate the membrane interface in a PI(3)P-dependent manner.Here, the finite difference Poisson-Boltzmann (FDPB) method has been used to calculate the energetic interactions of FYVE domains with phospholipid membranes. Based on the computational analysis, it is found that (1) recruitment to membranes is facilitated by non-specific electrostatic interactions that occur between basic residues on the domains and acidic phospholipids in the membrane, (2) the energetic analysis can quantitatively differentiate among the modes of membrane association proposed by the experimentally determined structures, (3) FDPB calculations predict energetically feasible models for the membrane-associated states of FYVE domains, (4) these models are consistent with the observation that conserved hydrophobic residues insert into the membrane interface, and (5) the calculations provide a molecular model for the hydrophobic partitioning: binding of PI(3)P significantly neutralizes positive potential in the region of the hydrophobic residues, which acts as an "electrostatic switch" by reducing the energetic barrier for membrane penetration. Finally, the computational results are extended to FYVE domains of unknown structure through the construction of high quality homology models for human FYVE sequences.