Outer Membrane Leaflet Research Articles

OsFBN7-OsKAS I module promotes formation of plastoglobules clusters in rice chloroplasts.

Plastoglobules (PGs) contiguous with the outer leaflets of thylakoid membranes regulate lipid metabolism, plastid developmental transitions, and responses to environmental stimuli. However, the function of OsFBN7, a PG-core fibrillin gene in rice, has not been elucidated. Using molecular genetics and physiobiochemical approaches, we observed that OsFBN7 overexpression promoted PG clustering in rice chloroplasts. OsFBN7 interacted with two KAS I enzymes, namely OsKAS Ia and OsKAS Ib, in rice chloroplasts. Lipidomic analysis of chloroplast subcompartments, including PGs in the OsFBN7 overexpression lines, confirmed that levels of diacylglycerol (DAG), a chloroplast lipid precursor and monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), the main chloroplast membrane lipids, were increased in PGs and chloroplasts. Furthermore, OsFBN7 enhanced the abundances of OsKAS Ia/Ib in planta and their stability under oxidative and heat stresses. In addition, RNA sequencing and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analyses showed that the expression of the DAG synthetase gene PAP1 and MGDG synthase gene MDG2 was upregulated by OsFBN7. In conclusion, this study proposes a new model in which OsFBN7 binds to OsKAS Ia/Ib in chloroplast and enhances their abundance and stability, thereby regulating the chloroplast and PG membrane lipids involved in the formation of PG clusters.

(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins II: Intercellular Transfer of Matter (Inheritance?) That Matters

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of the plasma membrane (PM) bilayer by covalent linkage to a typical glycolipid and expressed in all eukaryotic organisms so far studied. Lipolytic release from PMs into extracellular compartments and intercellular transfer are regarded as the main (patho)physiological roles exerted by GPI-APs. The intercellular transfer of GPI-APs relies on the complete GPI anchor and is mediated by extracellular vesicles such as microvesicles and exosomes and lipid-free homo- or heteromeric aggregates, and lipoprotein-like particles such as prostasomes and surfactant-like particles, or lipid-containing micelle-like complexes. In mammalian organisms, non-vesicular transfer is controlled by the distance between donor and acceptor cells/tissues; intrinsic conditions such as age, metabolic state, and stress; extrinsic factors such as GPI-binding proteins; hormones such as insulin; and drugs such as anti-diabetic sulfonylureas. It proceeds either “directly” upon close neighborhood or contact of donor and acceptor cells or “indirectly” as a consequence of the induced lipolytic release of GPI-APs from PMs. Those displace from the serum GPI-binding proteins GPI-APs, which have retained the complete anchor, and become assembled in aggregates or micelle-like complexes. Importantly, intercellular transfer of GPI-APs has been shown to induce specific phenotypes such as stimulation of lipid and glycogen synthesis, in cultured human adipocytes, blood cells, and induced pluripotent stem cells. As a consequence, intercellular transfer of GPI-APs should be regarded as non-genetic inheritance of (acquired) features between somatic cells which is based on the biogenesis and transmission of matter such as GPI-APs and “membrane landscapes”, rather than the replication and transmission of information such as DNA. Its operation in mammalian organisms remains to be clarified.

#3627 PHOSPHATIDYLSERINE EXPOSURE AND SHORTENED LIFE SPAN OF ERYTHROCYTES IN RENAL ANEMIA

Abstract Background and Aims The lifespan of erythrocytes in patients with renal anemia is shortened (∼70 days) compared to that of healthy erythrocytes (120 days), but the pathological mechanisms and causes are unclear. Senescent erythrocytes express phosphatidylserine (PS) on their outer membrane, which is a signal for being removed by macrophages. The PS localization in the cellular membrane is mainly regulated by two transporters: flippases and scramblases. Flippases, type Ⅳ P-type ATPases (P4-ATPases), maintain PS localization in the inner leaflet by transporting PS from the outer leaflet of the cell membrane. When scramblases are activated, they facilitate equal distribution of phospholipids between the inner and outer lipid bilayers, resulting in the exposure of PS on the outer leaflet. Recently, we have revealed that reduced flippase activity contributes to PS exposure in healthy senescent erythrocytes [1]. In this study, we examined the percentage of PS-exposed cells and the transporter activity in erythrocytes from renal anemia to elucidate the mechanism of the shortened lifespan. Method This study was approved by the Ethics Committee of Tokyo Women's Medical University (#3835, #4822R2). Erythrocytes from the renal anemic patients undergoing maintenance hemodialysis were separated with the centrifugation using Percoll-density gradient according to our previous report. Resultant heavy and light fractions were used as senescent and young erythrocytes, respectively. We analyzed the percentage of PS-exposed cells (n = 12), flippase/scramblase activities (n = 7), and the factors involved in those enzymatic activities such as intracellular K+ (n = 10) and ATP concentration (n = 9). We also measured protein 4.1a/b and HbA1c as an index of erythrocytes’ age. Results The cells from renal anemia were separated to heavy (senescent) and light (young) fractions, which was similar to the distributions previously performed on healthy erythrocytes. The PS-exposed erythrocytes in the heavy fraction were slightly more than those of the light fraction (p < 0.01, Wilcoxon test), and the flippase activity of the heavy fraction decreased more than that of the light fraction (p < 0.01, paired t-test). Intracellular K+ concentration decreased in the heavy fraction more than in the light fraction similar to the healthy senescent erythrocytes, but there was no statistical difference in the intracellular ATP concentration between the heavy and light fractions. The measurement of protein 4.1a/b and HbA1c in these fractions is still in progress. Conclusion Similar to healthy erythrocytes, reduced flippase activity contributes to PS exposure in senescent erythrocytes of renal anemia, maybe due to the decreased intracellular K+ concentration. This may occur in an earlier stage of senescence than the healthy ones, which may explain the mechanism of the shortened life span.

Characterization of an evolutionarily distinct bacterial ceramide kinase from Caulobacter crescentus

A common feature among nearly all gram-negative bacteria is the requirement for lipopolysaccharide (LPS) in the outer leaflet of the outer membrane. LPS provides structural integrity to the bacterial membrane, which aids bacteria in maintaining their shape and acts as a barrier from environmental stress and harmful substances such as detergents and antibiotics. Recent work has demonstrated that Caulobacter crescentus can survive without LPS due to the presence of the anionic sphingolipid ceramide-phosphoglycerate (CPG). Based on genetic evidence, we predicted that protein CpgB functions as a ceramide kinase and performs the first step in generating the phosphoglycerate head group. Here, we characterized the kinase activity of recombinantly expressed CpgB and demonstrated that it can phosphorylate ceramide to form ceramide 1-phosphate. The pH optimum for CpgB was 7.5, and the enzyme required Mg2+ as a cofactor. Mn2+, but no other divalent cations, could substitute for Mg2+. Under these conditions, the enzyme exhibited typical Michaelis-Menten kinetics with respect to NBD C6-ceramide (Km,app= 19.2± 5.5μM; Vmax,app= 2590± 230 pmol/min/mg enzyme) and ATP (Km,app= 0.29± 0.07mM; Vmax,app= 10,100± 996 pmol/min/mg enzyme). Phylogenetic analysis of CpgB revealed that CpgB belongs to a new class of ceramide kinases, which is distinct from its eukaryotic counterpart; furthermore, the pharmacological inhibitor of human ceramide kinase (NVP-231) had no effect on CpgB. The characterization of a new bacterial ceramide kinase opens avenues for understanding the structure and function of the various microbial phosphorylated sphingolipids.

TMEM16E regulates endothelial cell procoagulant activity and thrombosis.

Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid "scramblases," such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall-dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

Urtica dioica Leaf Infusion Enhances the Sensitivity of Triple-Negative Breast Cancer Cells to Cisplatin Treatment.

Urtica dioica (UD) has been widely used in traditional medicine due to its therapeutic benefits, including its anticancer effects. Natural compounds have a promising potential when used in combination with chemotherapeutic drugs. The present study explores the anticancer and anti-proliferative properties of UD tea in combination with cisplatin on MDA-MB-231 breast cancer cells in vitro. To elucidate the effect of this combination, a cell viability assay, Annexin V/PI dual staining, cell death ELISA, and Western blots were performed. The results showed that the combination of UD and cisplatin significantly decreased the proliferation of MDA-MB-231 cells in a dose- and time-dependent manner compared to each treatment alone. This was accompanied by an increase in two major hallmarks of apoptosis, the flipping of phosphatidylserine to the outer membrane leaflet and DNA fragmentation, as revealed by Annexin V/PI staining and cell death ELISA, respectively. DNA damage was also validated by the upregulation of the cleaved PARP protein as revealed by Western blot analysis. Finally, the increase in the Bax/Bcl-2 ratio further supported the apoptotic mechanism of death induced by this combination. Thus, a leaf infusion of Urtica dioica enhanced the sensitivity of an aggressive breast cancer cell line to cisplatin via the activation of apoptosis.

Artificial Cells for Cellular Behavior Mimicry Induced by Sphingomyelin Degradation.

Sphingomyelinase (SMase), a hydrolase of sphingomyelin (SM) enriched in the outer leaflet of the plasma membrane of mammalian cells, is closely associated with the onset and development of many diseases, but the specific mechanisms of SMase on the cell structure, function, and behavior are not yet fully understood due to the complexity of the cell structure. Artificial cells are minimal biological systems constructed from various molecular components designed to mimic cellular processes, behaviors, and structures, which are excellent models for studying biochemical reactions and dynamic changes in cell membranes. In this work, we presented an artificial cell model that mimics the lipid composition and content of the outer leaflet of mammalian plasma membranes for studying the effect of SMase on cell behavior. The results confirmed that the artificial cells can respond to SM degradation by producing ceramides that enrich and alter the membrane charge and permeability, thus inducing the budding and fission of the artificial cells. Thus, the artificial cells developed here provide a powerful tool to study the mechanism of action of cell membrane lipids on cell biological behavior, paving the way for further molecular mechanism studies.

(Patho)Physiology of Glycosylphosphatidylinositol-Anchored Proteins I: Localization at Plasma Membranes and Extracellular Compartments.

Glycosylphosphatidylinositol (GPI)-anchored proteins (APs) are anchored at the outer leaflet of plasma membranes (PMs) of all eukaryotic organisms studied so far by covalent linkage to a highly conserved glycolipid rather than a transmembrane domain. Since their first description, experimental data have been accumulating for the capability of GPI-APs to be released from PMs into the surrounding milieu. It became evident that this release results in distinct arrangements of GPI-APs which are compatible with the aqueous milieu upon loss of their GPI anchor by (proteolytic or lipolytic) cleavage or in the course of shielding of the full-length GPI anchor by incorporation into extracellular vesicles, lipoprotein-like particles and (lyso)phospholipid- and cholesterol-harboring micelle-like complexes or by association with GPI-binding proteins or/and other full-length GPI-APs. In mammalian organisms, the (patho)physiological roles of the released GPI-APs in the extracellular environment, such as blood and tissue cells, depend on the molecular mechanisms of their release as well as the cell types and tissues involved, and are controlled by their removal from circulation. This is accomplished by endocytic uptake by liver cells and/or degradation by GPI-specific phospholipase D in order to bypass potential unwanted effects of the released GPI-APs or their transfer from the releasing donor to acceptor cells (which will be reviewed in a forthcoming manuscript).

Neuraminidase causes endothelial phosphatidylserine externalization and ADAM17 activation: implications for shedding of insulin receptor and endothelial insulin resistance

Endothelial insulin resistance represents a causal factor in the pathogenesis of type 2 diabetes (T2D) and vascular disease, thus underscoring the need to identify molecular mechanisms underlying defects in endothelial insulin signaling. We previously showed that a disintegrin and metalloproteinase-17 (ADAM17) is increased while insulin receptor α-subunit (IRα) is decreased in the vasculature of patients with T2D, leading to impaired insulin-induced vasodilation. We also showed that ADAM17 is a sheddase that targets IRα. Externalization of phosphatidylserine (PS) from the inner to the outer leaflet of cell membranes has been proposed as a pivotal step in the activation of ADAM17. However, whether PS externalization increases ADAM17 sheddase activity and, consequently, shedding of endothelial IRα in T2D is not known. Herein, we hypothesized that neuraminidase (Neu), an enzyme we showed is upregulated in diabetes, leads to PS externalization via activation of Ca2+ influx and subsequent induction of ADAM17 sheddase activity. All reported differences are significant at P<0.05. In support of this hypothesis, we report that Neu and ADAM17 activities are increased in plasma of individuals with T2D, relative to non-T2D subjects. In experiments using cultured endothelial cells, we show that exposure to Neu increases Ca2+ influx and ADAM17 activity. We also find that augmenting endothelial Ca 2+ influx using a calcium ionophore (A23187) increases adhesion of lactadherin (a peptide that binds externalized PS) to the surface of endothelial cells, as does treatment with the ADAM17 activator, PMA, which also reduces IRα on the cells. Importantly, overexpression of ADAM17 in endothelial cells reduces IRα in cell lysates and this coincides with a tendency towards increased IRα in the cell culture supernatant, indicative of protein shedding. Collectively, these findings suggest that Neu causes Ca 2+ influx in endothelial cells, favoring PS externalization and, consequently, increased ADAM17 activity and shedding of IRα. Hence, inhibition of Neu and ADAM17 sheddase activities should be considered as novel potential strategies to improve endothelial insulin signaling in T2D. R01HL151384 (to LAML and JP), R01HL153264 (to LAML and JP), and R01HL137769 (to JP). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phentolamine Significantly Enhances Macrolide Antibiotic Antibacterial Activity against MDR Gram-Negative Bacteria.

Multidrug-resistant (MDR) Gram-negative bacterial infections have limited treatment options due to the impermeability of the outer membrane. New therapeutic strategies or agents are urgently needed, and combination therapies using existing antibiotics are a potentially effective means to treat these infections. In this study, we examined whether phentolamine can enhance the antibacterial activity of macrolide antibiotics against Gram-negative bacteria and investigated its mechanism of action. Synergistic effects between phentolamine and macrolide antibiotics were evaluated by checkerboard and time-kill assays and in vivo using a Galleria mellonella infection model. We utilized a combination of biochemical tests (outer membrane permeability, ATP synthesis, ΔpH gradient measurements, and EtBr accumulation assays) with scanning electron microscopy to clarify the mechanism of phentolamine enhancement of macrolide antibacterial activity against Escherichia coli. In vitro tests of phentolamine combined with the macrolide antibiotics erythromycin, clarithromycin, and azithromycin indicated a synergistic action against E. coli test strains. The fractional concentration inhibitory indices (FICI) of 0.375 and 0.5 indicated a synergic effect that was consistent with kinetic time-kill assays. This synergy was also seen for Salmonella typhimurium, Klebsiella pneumoniae, and Actinobacter baumannii but not Pseudomonas aeruginosa. Similarly, a phentolamine/erythromycin combination displayed significant synergistic effects in vivo in the G. mellonella model. Phentolamine added singly to bacterial cells also resulted in direct outer membrane damage and was able to dissipate and uncouple membrane proton motive force from ATP synthesis that, resulted in enhanced cytoplasmic antibiotic accumulation via reduced efflux pump activity. Phentolamine potentiates macrolide antibiotic activity via reducing efflux pump activity and direct damage to the outer membrane leaflet of Gram-negative bacteria both in vitro and in vivo.

Abstract 1272: Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) has risen to become the 3rd leading cause of cancer related deaths in the United States with a 5-year survival rate of 11% and a median survival of just 4-6 months. PDA is notoriously chemo-resistant and has shown little to no sensitivity to the current immune checkpoint blockade therapy. A novel marker for the tumor microenvironment is phosphatidylserine, first characterized as a selective marker of tumor vasculature. Phosphatidylserine (PS) is a phospholipid exposed on the outer leaflet of the plasma membrane of tumor associated endothelial cells, apoptotic tumor cells, and some viable tumor cells, where it functions to suppress the immune response. Exposed PS drives immunosuppression by binding to PS receptors expressed on tumor-infiltrating macrophages and dendritic cells. To this point, PS has been targeted with antibodies, such as Bavituximab, that have shown excellent specificity for tumor vasculature and an immune stimulatory environment evidence by polarization of macrophages to a pro-inflammatory M1-like phenotype, a reduction in myeloid-derived suppressor cells, enhanced maturation of dendritic cells and increased primed T cell activity. We have advanced this concept by developing the next generation of PS targeting agent, a fusion protein (betabody) that exploits the PS-binding domain of β2-glycoprotein 1 (β2GP1), an abundant serum protein that binds PS. Betabodies were constructed by fusing domain V of β2GP1 to the Fc of an IgG2a. Betabodies are smaller than full length antibodies (~85 kDa vs 150 kDa), have good affinity for PS (~1 nM) and bind directly to PS. We have demonstrated that betabodies bind specifically to externalized PS through ELISA, ICC and flow cytometry. We established that betabodies localize robustly and specifically to the tumor microenvironment through in vivo localization experiments in tumor-bearing mice. Further studies will exploit the specificity of betabodies to deliver novel microtubule-disrupting agents to exposed PS in the microenvironment of PDA. Because betabodies are internalized inefficiently, therapeutic payloads will be linked to betabodies via enzyme cleavable linkers. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors. In addition, similarly to PS targeting antibodies, preliminary data in vitro has shown that treatment of both bone marrow derived macrophages and Raw 264.7 with the unconjugated betabodies repolarize M2 macrophages to a M1 phenotype. The mechanism of action has not yet been fully elucidated; however, it may involve the interaction between the β2GP1 domains and endogenous LPS. This first-in-class tumor targeting therapeutic has potential to provide efficacy in therapy-resistant pancreatic tumors via targeted drug delivery as well as modulation of the immuno-microenvironment like its PS targeting predecessors. Citation Format: Natalie Zara Phinney, Xianming Huang, Mary Lynn Trawick, Kevin Pinney, Rolf Brekken. Aberrantly exposed phosphatidylserine as a drug delivery target in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1272.

Highly pathogenic natural monoclonal antibody B4-IgM recognizes a post-translational modification comprised of acetylated N-terminal methionine followed by aspartic or glutamic acid

The natural monoclonal antibody B4-IgM recognizes murine annexin 4 (mAn4) and exacerbates ischemia-reperfusion injury in many mouse models. During apoptosis, the intracellular mAn4 protein translocates to the membrane surface, remaining attached to the outer membrane leaflet where it is recognized by the anti-mAn4 B4-IgM antibody. B4-IgM does not recognize human annexin 4 (hAn4). However, the B4-IgM antibody epitope was detected by Western blot of unknown human proteins and by flow cytometry on all studied human cell lines undergoing apoptosis and on a minor subset of healthy cells. The B4-IgM antibody also recognizes the epitope on necrotic cells in cytoplasmic proteins, apparently entering through pores large enough to allow natural antibodies to penetrate the cells and bind to the epitope expressed on self-proteins. Using proteomics and site-directed mutagenesis, we found that B4-IgM binds to an epitope with post-translationally modified acetylated N-terminal methionine, followed by either glutamic or aspartic acid. The epitope is not induced by apoptosis or injury because this modification can also occur during protein translation. This finding reveals an additional novel mechanism whereby injured cells are detected by natural antibodies that initiate pathogenic complement activation through the recognition of epitopes that are shared across multiple proteins found in variable cell lines.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes

AbstractPhospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

Epitope Imprinting of Phospholipids by Oriented Assembly at the Oil/Water Interface for the Selective Recognition of Plasma Membranes.

Phospholipids, as fundamental building blocks of the cell membrane, play important roles for molecule transportation, cell recognition, etc. However, due to the structural diversity and amphipathic nature, there are few methods for the specific recognition of lipids as compared to other biomolecules such as proteins and glycans. Herein, we developed a molecular imprinting strategy for controllable imprinting toward the polar head of phospholipid exposed on the surface of cellular membranes for recognition. Phosphatidylserine, as unique lipid on the outer membrane leaflet of exosome and also hallmark for cell apoptosis, was imprinted with the developed method. The phosphatidylserine imprinted materials showed high efficiency and specific targeting capability not only for apoptotic cell imaging but also for the isolation of exosomes. Collectively, the synthesized molecularly imprinted materials have great potential for selective plasma membrane recognition for targeted drug delivery and biomarker discovery.

The Direct Anti-Virulence but Not Bactericidal Activity of Human Neutrophil Elastase against Moraxella catarrhalis.

Neutrophil elastase (NE) contributes to innate antibacterial defense at both the intracellular (phagocytosis) and extracellular (degranulation, NETosis) levels. Moraxella catarrhalis, a human respiratory pathogen, can exist in an inflammatory milieu which contains NE. No data are available on the action of NE against M. catarrhalis or on the counteraction of NE-dependent host defenses by this pathogen. Using time-kill assays we found that bacteria are able to survive and replicate in the presence of NE. Transmission electron microscopy and flow cytometry studies with NE-treated bacteria revealed that while NE admittedly destabilizes the outer membrane leaflet, it does not cause cytoplasmic membrane rupture, suggesting that the enzyme does not target components that are essential for cell integrity. Using LC-MS/MS spectroscopy we determined that NE cleaved at least three virulent surface proteins in outer membrane vesicles (OMVs) of M. catarrhalis, including OMP CD, McaP, and TbpA. The cleavage of OMP CD contributes to the significant decrease in resistance to serum complement in the complement-resistant strain Mc6. The cleavage of McaP did not cause any sensitization to erythromycin nor did NE disturb its drug action. Identifying NE as a novel but subtle anti-virulence agent together with its extracellularly not-efficient bactericidal activity against M. catarrhalis may facilitate the pathogen's existence in the airways under inflammation.

Exploring the Role of PI3P in Platelets: Insights from a Novel External PI3P Pool

Phosphoinositides (PIs) play a crucial role in regulating intracellular signaling, actin cytoskeleton rearrangements, and membrane trafficking by binding to specific domains of effector proteins. They are primarily found in the membrane leaflets facing the cytosol. Our study demonstrates the presence of a pool of phosphatidylinositol 3-monophosphate (PI3P) in the outer leaflet of the plasma membrane of resting human and mouse platelets. This pool of PI3P is accessible to exogenous recombinant myotubularin 3-phosphatase and ABH phospholipase. Mouse platelets with loss of function of class III PI 3-kinase and class II PI 3-kinase α have a decreased level of external PI3P, suggesting a contribution of these kinases to this pool of PI3P. After injection in mouse, or incubation ex vivo in human blood, PI3P-binding proteins decorated the platelet surface as well as α-granules. Upon activation, these platelets were able to secrete the PI3P-binding proteins. These data sheds light on a previously unknown external pool of PI3P in the platelet plasma membrane that recognizes PI3P-binding proteins, leading to their uptake towards α-granules. This study raises questions about the potential function of this external PI3P in the communication of platelets with the extracellular environment, and its possible role in eliminating proteins from the plasma.

Transfer of Proteins from Cultured Human Adipose to Blood Cells and Induction of Anabolic Phenotype Are Controlled by Serum, Insulin and Sulfonylurea Drugs.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are anchored at the outer leaflet of eukaryotic plasma membranes (PMs) only by carboxy-terminal covalently coupled GPI. GPI-APs are known to be released from the surface of donor cells in response to insulin and antidiabetic sulfonylureas (SUs) by lipolytic cleavage of the GPI or upon metabolic derangement as full-length GPI-APs with the complete GPI attached. Full-length GPI-APs become removed from extracellular compartments by binding to serum proteins, such as GPI-specific phospholipase D (GPLD1), or insertion into the PMs of acceptor cells. Here, the interplay between the lipolytic release and intercellular transfer of GPI-APs and its potential functional impact was studied using transwell co-culture with human adipocytes as insulin-/SU-responsive donor cells and GPI-deficient erythroleukemia as acceptor cells (ELCs). Measurement of the transfer as the expression of full-length GPI-APs at the ELC PMs by their microfluidic chip-based sensing with GPI-binding α-toxin and GPI-APs antibodies and of the ELC anabolic state as glycogen synthesis upon incubation with insulin, SUs and serum yielded the following results: (i) Loss of GPI-APs from the PM upon termination of their transfer and decline of glycogen synthesis in ELCs, as well as prolongation of the PM expression of transferred GPI-APs upon inhibition of their endocytosis and upregulated glycogen synthesis follow similar time courses. (ii) Insulin and SUs inhibit both GPI-AP transfer and glycogen synthesis upregulation in a concentration-dependent fashion, with the efficacies of the SUs increasing with their blood glucose-lowering activity. (iii) Serum from rats eliminates insulin- and SU-inhibition of both GPI-APs' transfer and glycogen synthesis in a volume-dependent fashion, with the potency increasing with their metabolic derangement. (iv) In rat serum, full-length GPI-APs bind to proteins, among them (inhibited) GPLD1, with the efficacy increasing with the metabolic derangement. (v) GPI-APs are displaced from serum proteins by synthetic phosphoinositolglycans and then transferred to ELCs with accompanying stimulation of glycogen synthesis, each with efficacies increasing with their structural similarity to the GPI glycan core. Thus, both insulin and SUs either block or foster transfer when serum proteins are depleted of or loaded with full-length GPI-APs, respectively, i.e., in the normal or metabolically deranged state. The transfer of the anabolic state from somatic to blood cells over long distance and its "indirect" complex control by insulin, SUs and serum proteins support the (patho)physiological relevance of the intercellular transfer of GPI-APs.

A trimeric coiled-coil motif binds bacterial lipopolysaccharides with picomolar affinity.

α-helical coiled-coils are ubiquitous protein structures in all living organisms. For decades, modified coiled-coils sequences have been used in biotechnology, vaccine development, and biochemical research to induce protein oligomerization, and form self-assembled protein scaffolds. A prominent model for the versatility of coiled-coil sequences is a peptide derived from the yeast transcription factor, GCN4. In this work, we show that its trimeric variant, GCN4-pII, binds bacterial lipopolysaccharides (LPS) from different bacterial species with picomolar affinity. LPS molecules are highly immunogenic, toxic glycolipids that comprise the outer leaflet of the outer membrane of Gram-negative bacteria. Using scattering techniques and electron microscopy, we show how GCN4-pII breaks down LPS micelles in solution. Our findings suggest that the GCN4-pII peptide and derivatives thereof could be used for novel LPS detection and removal solutions with high relevance to the production and quality control of biopharmaceuticals and other biomedical products, where even minuscule amounts of residual LPS can be lethal.

Probing the role of RNA in prion protein and prion fibril recognition.

The conversion of the cellular form of the prion protein PrPC to the misfolded and aggregated form PrPSc is the key pathological event in prion diseases, yet the mechanism of the conformational change remains unknown. Evidence suggests that prion protein misfolding initiates on the cell surface, where the prion protein is GPI anchored to the outer leaflet of the plasma membrane. Compelling results document that extracellularly located nucleic acids scaffold prion protein aggregation; however, whether nucleic acids interact directly with the monomer prion protein, a misfolded intermediate, or PrPSc fibrils remains unknown. Recent studies indicate upon RNA binding, a pincer-like structure is formed between the N-terminus and a-helix 1 of the prion protein that traps the RNA and induces misfolding of the helix. However, it is unknown how the initial misfolding of a-helix 1 affects the other helices. To fill this gap, we aim to examine the changes in protein and fibril connectivity as well as identify key residues involved in RNA mediated aggregation of the prion protein. We used structural bioinformatics techniques to model and examine the connectivity of the prion protein and prion fibrils when bound to RNA. Our results suggest that the binding of RNA to a-helix 1 modulates the long-distance coupling to other helices. We will also probe the modulatory effects of RNA on the interactions between prion protein and fibrils. We will use these calculations to infer the role of RNA on prion protein misfolding and protein-fibril recognition.

Exploring membrane binding targets of tau oligomers on phase-separated lipid bilayers using multiscale MD simulations.

Self-aggregation of tau has been linked to the onset of Alzheimer's. Recent studies indicate that the disordered tau oligomers are more neurotoxic than the ordered tau fibrils in the intracellular neurofibrillary tangle. Details of tau oligomer interactions with the lipid nanodomains in phase-separated lipid bilayers (raft membranes), a model of neuronal membranes, are unknown. Using coarse-grained (CG) and all-atom (AA) MD simulations, a tau-K18 monomer containing the microtubule-binding domain (R1-R2-R3-R4) and its disordered oligomers were simulated. By mixing phosphatidylcholine, cholesterol, ganglioside (GM1), and phosphatidylserine (PS) lipids in water, raft membranes containing lipid nanodomains, liquid-ordered (Lo), liquid-disordered (Ld), and Lo/Ld boundary (Lod) on both lipid leaflets, as well as PS- or GM1-cluster on one lipid leaflet, were simulated. Lipid-binding kinetics and energetics of tau-K18 oligomers to these nanodomains were investigated. Within 20 μs, tau-K18 oligomers bound to the Lod domains in membranes without PS or GM1. In contrast, exclusive binding to PS- or GM1-clusters in membranes containing PS or GM1 occurred in less than 6 μs. After a CG to AA transformation and a 200 ns-AA simulation, disruptions of lipid chain order and beta-sheet formation on raft membranes containing membrane-bound tau-K18 oligomers were found. By mutating three hydrophobic residues to anionic residues at the R2 and R3 domain regions of tau-K18, the mutated tau-K18 oligomers bound much weaker to the PS- or GM1-cluster domains than the wild type did, suggesting the importance of electrostatic interactions in the tau-lipid interactions. Since PS and GM1 are found in the inner and outer leaflets of neuronal membranes, respectively, our results suggest that the PS- and GM1-clusters are leaflet-specific targets while the Lod domains are non-specific targets in membrane-associated tauopathies in Alzheimer's. (Supported by NSF [OAC-153159] and Welch Foundation [W-2057-2021-327].)