Presence Of Phospholipid Vesicles Research Articles

Contribution of the Residues 488-490 on the Factor (F)VIII A2 Domain and the Residue 2239 on the FVIII C2 Domain for the Interaction between FVIII and Protein S

Factor (F)VIII functions as a cofactor in the Xase complex responsible for phospholipid (PL) surface-dependent conversion of FX to activated FX (FXa) by FIXa. Protein S (PS), a vitamin K-dependent anticoagulant protein, functions as a cofactor of activated protein C (APC) that inactivates FVIIIa and FVa. We have previously reported a new regulatory mechanism on coagulation that PS directly impaired the Xase complex by competing the FVIIIa-FIXa interaction (Takeyama, Br J Haematol. 2008), and identified at least two PS-interactive sites that were the 488-490 residues on FVIII A2 domain (Takeyama, Thromb Haemost. 2009) and the 2239 residue on FVIII C2 domain (Furukawa, ASH 2016). In the present study, to investigate the contribution of binding sites on FVIII A2 and C2 domains for the interaction between FVIII and PS, three recombinant B-domainless FVIII mutants substituted to alanine were expressed by BHK cell, such as the mutant of the 488-490 residues (S488A/R489A/R490A), the mutant of the 2239 residue (K2239A), and the mutant of both residues (S488A/R489A/R490A/K2239A), subsequently compared interaction with PS of the three mutants with wild-type (wt-) FVIII. First, we performed surface plasmon resonance-based assays to compare the direct binding of each FVIII mutant to PS. When PS was immobilized on the sensor chip and bound to the wt-FVIII and each FVIII mutant as an analyte, the K d value of wt-FVIII was 3.5 nM, while those of S488A/R489A/R490A, K2239A, and S488A/R489A/R490A/K2239A mutants were 43.7 nM, 6.7 nM, and 44.7 nM, respectively. These results demonstrated that the residues 488-490 and 2239 contributed to PS-interactive sites as described in the previous reports. Next, the effect of PS on the directly reduction of FVIIIa function was evaluated using the FXa generation assay with the three FVIII mutants and compared with wt-FVIII. After each FVIII mutant (1 nM) was reacted with PS (0-500 nM) in the presence of PL vesicles (20 µM) for 30 min, FVIII was activated by thrombin (30 nM), followed by the reaction with FIXa (40 nM) and FX (300 nM) for 1 min. Generated FXa in wt-FVIII was reduced in the presence of PS dose-dependently and was reduced to 7.7% at 125 nM of PS, compared to that in the absence of PS. However, generated FXa in K2239A, S488A/R489A/R490A, and S488A/R489A/R490A/K2239A were mildly reduced to 23.1 %, 23.6 %, and 29.2 % even at 500 nM of PS, respectively, compared to those in the absence of PS. Finally, to investigate the contribution of these FVIII and PS binding sites when PS plays as a cofactor of APC in FVIIIa inactivation, FXa generation assays were measured in each FVIII mutant in the presence of PS (150 nM) and APC (0-10 nM). In the presence of PS, generated FXa in wt-FVIII and each FVIII mutant were decreased in APC dose-dependently. At the 10 nM of APC, generated FXa in the wt-FVIII and K2239A were reduced to 48.1 % and 40.6 %, respectively, in comparison to the absence of APC, while generated FXa in S488A/R489A/R490A and S488A/R489A/R490A/K2239A mutants were mildly reduced to 65.8 % and 69.0 %, respectively. These findings suggested that the contribution of the 488-490 residues on the FVIII A2 domain was particularly great for the FVIII-PS interaction between the two PS binding sites identified on the FVIII A2 and C2 domains. In addition, the coexistence of the K2239A mutation on the FVIII C2 domain with S488A/R489A/R490A mutation further reduced the direct FVIII-PS interaction, suggesting that K2239A mutation further reduced the inhibitory effect of FVIII function by PS. On the other hand, mainly not the 2239 residue on the FVIII C2 domain but the 488-490 residues on the FVIII A2 domain were thought to affect the PS function as a cofactor in FVIIIa inactivation by APC. Overall, our data indicated that the 488-490 residues on the FVIII A2 domain were key residues for the FVIII-PS interaction including FVIIIa inactivation by APC, and the 2239 residue affected partially for only the FVIII-PS direct interaction.

Metastable intermediate during hIAPP aggregation catalyzed by membranes as detected with 2D IR spectroscopy.

The aggregation of human islet amyloid polypeptide (hIAPP) into amyloid fibrils involves formation of oligomeric intermediates that are thought to be the cytotoxic species responsible for β-cell dysfunction in type 2 diabetes. hIAPP oligomers permeating or disrupting the cellular membrane may be one mechanism of toxicity and so measuring the structural kinetics of aggregation in the presence of membranes is of much interest. In this study, we use 2D IR spectroscopy and 13C18O isotope labeling to study the secondary structure of the oligomeric intermediates formed in solution and in the presence of phospholipid vesicles at sites L12A13, L16V17, G24A25 and V32G33. Pairs of labels monitor the couplings between associated polypeptides and the dihedral angles between adjacent residues. In solution, the L12A13 residues form an oligomeric β-sheet in addition to an α-helix whereas with the phospholipid vesicles they are α-helical throughout the aggregation process. In both solution and with DOPC vesicles, L16V17 and V32G33 have disordered structures until fibrils are formed. Similarly, under both conditions, G24A25 exhibits 3-state kinetics, created by an oligomeric intermediate with a well-defined β-sheet structure. Amyloid fibril formation is often thought to involve intermediates with exceedingly low populations that are difficult to detect experimentally. These experiments establish that amyloid fibril formation of hIAPP when catalyzed by membranes includes a metastable intermediate and that this intermediate has a similar structure at G24A25 in the FGAIL region as the corresponding intermediate in solution, thought to be the toxic species.

Inhibition of Factor IXa By Protein S: Critical Function of the Protein S SHBG-like Domain

Background and Approach -Protein S (PS) is a vitamin K-dependent anticoagulant. PS deficiency can have severe-even deadly-outcomes, such as deep vein thrombosis, arterial thrombosis (stroke, heart attack), and pulmonary embolism. We have demonstrated a direct inhibition of FIXa by PS independent of Protein C, in the absence and presence of FVIIIa. However, the binding sites for FIXa within PS have not been identified; such identification is a necessary first step to enlisting PS in the prevention of life-threatening thrombosis. Thus, we used an in silico docking approach with the three dimensional structure of SHBG domain of PS that predicted the involvement of SHBG domain/s to mediate FIXa binding. The SHBG is composed of mainly two laminin LG-type domains, LG1 and LG2.Aim To determine whether the LG1, LG2, or LG1+2 domains of PS can bind and inhibit FIXa.Methods- Human PS LG1, LG2, and LG1+LG2 domains were cloned into the GST tagging pGEXGP1 expression vector, and the expressed polypeptides were purified with glutathione Sepharose. Binding of the LG1+2 construct to FIXa was measured by anisotropy. Association of LG1+2 with FIXa was also assessed by co-immunoprecipitation from plasma. We also measured the inhibitory effect of the LG constructs on FIXa activation of FX in the presence and absence of FVIIIa and phospholipid vesicles (PS/PC). Finally, we measured plasma thrombin generation in PS-deficient plasma supplemented with the LG domain constructs.Results- We measured the anisotropy of active site-labeled FIXa (DEGR-IXa) in the presence of phosphatidylserine/phosphatidylcholine vesicles and increasing concentrations of LG1+2 protein. A binding Kd of ~ 50 nM was obtained from these anisotropy measurements, which was comparable to the binding Kd of PS to FIXa (Kd ~ 40 nM). We further confirmed the association between LG1+2 and FIXa by co-immunoprecipitation. PS-deficient plasma was supplemented with either LG1+2 or intact PS and incubated with anti-PS antibody, followed by immunoblotting of the immunoprecipitates with a FIXa-specific antibody; both PS and LG1+2 protein had associated with FIXa. Next, we measured the proteolytic activation of FX by FIXa in the presence of the LG constructs; FX activation was quantitated by cleavage of a synthetic FX substrate (S-2765) in the presence of phospholipid vesicles. The LG1+2 construct reduced the rate of FX activation by FIX to ~ 54%, and the rate was further reduced to 69% when FVIIIa was complexed with FIXa; these reduced rates agreed with our previous measurements using intact PS. However, the separate LG1 and LG2 domains reduced the rate of FX activation by FIXa by only 35 % and 29%, respectively. Plasma thrombin generation with a limiting amount of Tissue Factor also supported the results obtained from the FX activation assay as described above. The effects of PS and the LG1+2 construct on thrombin generation were the same (Figure 1).Conclusion- Binding measurements, proteolytic studies, and plasma thrombin generation assays indicate that the LG1+2 domain of PS is sufficient to inhibit FIXa. The LG1 and LG2 region of the PS SHBG domain are the critical contact regions involved in FIXa inhibition. Hence, our study suggests that PS deficiencies could be treated with a small laminin G-like domain of PS instead of the much larger intact PS protein. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Molecular Interaction Site on Procoagulant Skeletal Muscle Myosin for Factor Xa-Dependent Prothrombin Activation

Skeletal muscle myosin (SkM) is a muscle protein consisting of a dimer of heterotrimers, each trimer comprising a regulatory light chain (RLC), an essential light chain (ELC), and a heavy chain (HC). Recently it was discovered that SkM has potent procoagulant and prothrombotic activity (Deguchi H, et al, Blood. 2016;128:1870-1878). Mechanistic studies showed that SkM's potent prothrombotic activity involved enhancing thrombin generation due to SkM's ability to bind coagulation factors Xa and Va which accelerates prothrombin activation. However, detailed molecular mechanisms for SkM's binding of these coagulation factors have not been described. Since a well-known myosin inhibitor, trifluoperazine (TFP), inhibited SkM's procoagulant activity and since this inhibitor binds to the ELC in SkM's "neck" region which connects the HC head region to the HC tail (Figure, panel A), we hypothesized that SkM's TFP binding region on the ELC in the neck region directly contributes to SkM's procoagulant activity. To identify potential binding site(s) on SkM for factors Xa and Va, 22 peptides representing the neck region's RLC, ELC, and HC were screened for inhibition of SkM-supported prothrombin activation by purified factor Xa, factor Va, and calcium ions. These peptides contained 25-40 residues and overlapped by approximately 5-10 amino acids. Peptides ELC109-138 and ELC129-159, corresponding to amino acid residues 109-138 and 129-159 of the ELC, inhibited SkM-supported prothrombin activation at 100 μM, whereas their partially overlapping neighboring peptides, ELC99-122 and ELC149-173, did not. Three HC peptides (peptides HC781-810, HC796-835, HC815-854) and one RLC peptide (RLC133-162) inhibited SkM-supported prothrombin activation at 100 μM, and each was also inhibitory, to varying degrees, when assayed at 5 μM. Dose-dependency inhibition assays gave IC50 values (50% inhibition of activity) for the peptides HC781-810, HC796-835, HC815-854, and RLC133-162 of 64, 1.2, 2.3 and 26 μM. Peptides HC781-810 and HC815-854 also inhibited prothrombin activation in the absence of myosin but in the presence of phospholipid vesicles containing 20 % phosphatidylserine (IC50 = 7.5 and 104 μM, respectively). In contrast, the strong inhibitory effects of peptides HC796-835, RLC133-162, ELC109-138 and ELC129-159 seen in the presence of myosin were not at all apparent in the presence of phospholipid-supported prothrombin activation when myosin was absent. This suggests that peptides HC796-835, RLC133-162, ELC109-138 and ELC129-159 specifically inhibit SkM-supported prothrombin activation. The 19 synthetic peptides representing the SkM neck region were also screened at 25 µM (final) for their inhibition of recalcification-induced thrombin generation in human plasma which contains significant circulating levels of SkM. Among the 19 peptides tested, HC796-835 and HC815-854 significantly inhibited thrombin generation when screened at 25 µM in plasma. Immobilized peptide HC796-835 showed direct binding of purified factor Xa with apparent Kd of 1.4 μM. This very potent inhibitory peptide, HC796-835, exhibited 50% inhibition of SkM-enhanced prothrombin activation at 1.2 μM, indicating that this peptide's sequence provides a factor Xa binding site on SkM which contributes to its inhibitory action. More specifically, an overlapping peptide containing amino acid residues 815-835 inhibited SkM-enhanced prothrombin activation by factors Xa and Va while a peptide comprising residues 796-811 did not. These studies suggest that residues 815-835 of SkM's HC are responsible for directly binding factor Xa and implies that this binding is responsible for SkM's procoagulant activity (Figure, panel B). In summary, we identified human SkM peptides which specifically blocked SkM-enhanced thrombin generation but not phospholipid-stimulated prothrombin activation in purified reaction mixtures and which inhibited blood clotting in plasma. The most potent anticoagulant HC peptide also directly binds purified factor Xa. These findings strongly suggest that the neck region of SkM, as defined by these inhibitory peptides (Figure, panel B), provides a phospholipid-independent procoagulant surface for thrombin generation that, depending on the in vivo physiologic context, may contribute to either hemostasis or thrombosis. Figure Disclosures No relevant conflicts of interest to declare.

Markedly Reduced Expression of Annexin A2 Is Associated with Hypofibrinolysis and Provoked and Unprovoked Venous Thromboembolism

Introduction: Recent evidence indicates that plasma hypofibrinolysis, as measured by clot lysis times (CLT) at or above the 90th percentile, can double the risk of venous thromboembolism (VTE). To our knowledge, there are no studies on the role of cell surface fibrinolysis in thrombosis. Annexin A2 (A2) is a calcium dependent, phospholipid binding protein that forms a heterotetramer with S100A10 (A2-S100A10)2 on the surface of endothelial cells (ECs). This complex serves as a co-receptor for plasminogen and tissue plasminogen activator (tPA), and increases the catalytic efficiency of tPA-dependent cell surface plasmin generation by 60-fold. In mouse studies, global knockout of the annexin A2 gene (Anxa2) is associated with fibrin accumulation and impaired clearance of arterial thrombi. In addition, there are several examples of the regulatory role of A2 in fibrinolysis in human diseases such as antiphospholipid antibody syndrome, cerebral venous thrombosis, and sickle cell disease. In the current study, we aimed to explore the potential role of the A2 system and cell surface based fibrinolysis in the development of VTE. Methods: Study subjects included patients 18-65 years old with history of VTE and healthy controls. Subjects were classified as having provoked or unprovoked VTE based on the presence or absence of identifiable environmental risk factors. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected in EDTA tubes, and used as a surrogate for ECs. Assays performed on PBMCs included determination of the rate of tPA-dependent cell surface plasmin generation using a fluorogenic substrate and analysis of CLT in the presence of phospholipid vesicles. Because A2 accounts for approximately 50% of cell surface based plasmin generation on both ECs and PBMCs, we analyzed total A2 protein expression relative to GAPDH in whole cell lysates of PBMCs using semi-quantitative western blotting. Additional assays included quantitative RT-PCR and ANXA2 gene sequencing. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparisons test. Results: Overall, 116 subjects with VTE (76 with provoked and 40 with unprovoked VTE) and 87 healthy controls were studied between September 2010 and May 2019. Plasma based clot lysis assays revealed that the mean CLT for subjects with VTE was significantly longer than that for healthy controls (37.5 versus 30.7 minutes, p=0.0001). Additionally, the mean rate of cell surface plasmin generation was significantly reduced in subjects with VTE as compared with healthy controls (0.33 RFU/min2 versus 0.49 RFU/min2, p=0.0021). Moreover, none of the 60 healthy controls (0%), but 18 of the 107 subjects with thrombosis (17%) had cell surface plasmin generating capacity in the lowest 10th percentile. In protein expression studies, we observed that none of the 21 healthy controls (0%), but 5 of 41 subjects with thrombosis (12%) had A2 expression in the lowest 10th percentile for the group; 4 of 18 (22%) of those with unprovoked VTE and 1 of 23 (4%) of those with provoked VTE fell into the lowest decile for protein expression. For plasmin generating capacity, 8 of 36 (22%) of subjects with unprovoked VTE and 10 of 71 (14%) of those with provoked VTE occupied the lowest decile. Probing of western blots of samples obtained on two separate occasions several months apart with A2 epitope-specific antibodies revealed abnormalities within either the tPA binding N-terminal tail or C-terminal core domain in proximity to the plasminogen binding site. Neither quantitative RT-PCR nor ANXA2 gene sequencing of selected samples revealed abnormalities in either mRNA or genomic DNA that could explain the reduced A2 expression. Conclusion: These data confirm findings previously reported by Lisman that plasma hypofibrinolysis is associated with VTE and may represent an independent risk factor for VTE. Additionally, we demonstrate for the first time that impaired cell surface based fibrinolysis and aberrations in A2 protein expression are associated with both provoked and unprovoked VTE, and may represent a novel risk factor for thrombosis. Possible explanations for reduced A2 expression include dysfunctional translation of mRNA into protein or post-translational proteolysis of the translated protein. Compromise of the A2-based fibrinolytic system may represent a previously unrecognized contributor to thrombophilia in VTE. Disclosures Ruisi: BMY: Equity Ownership; EMD, subsidiary of Merck KGaA: Employment. De Sancho:Apellis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Phospholipid-Independent Activity of Fviiia Mimetic Bispecific Antibodies in Plasma

Introduction: An important coagulation regulatory mechanism is localization of clotting complexes to exposed phosphatidylserine (PS) on cell surfaces. All components of the intrinsic tenase complex (factor (F)VIIIa, FIXa, and FX) bind to PS. FVIIIa mimetic bispecific antibodies are drugs in development for hemophilia A that aim to mimic the cofactor function of FVIIIa by bringing together FIXa and FX to generate FXa. However, these antibodies differ from FVIII in many ways including no requirement of activation and a lack of direct PS binding. Emicizumab is a bispecific antibody currently on the market for hemophilia A patients with inhibitors. It binds to factor FIX, FIXa, FX, and FXa with micromolar affinities in solution. Previously, we have shown that in-house preparations of sequence-identical emicizumab (SI-Emi) showed similar weak affinities to its antigens and similar in vitro activity to published emicizumab results by one-stage clotting, chromogenic FXa generation, and thrombin generation. However, in chromogenic FXa generation using antibody concentrations in the range of the mean steady state plasma concentration of patients on emicizumab prophylaxis [~360 nM, (Oldenburg, et al., NEJM 2017)], SI-Emi maintained 28% of its activity even in the absence of PS-containing phospholipid vesicles. Another FVIIIa mimetic antibody, BS-027125, was discovered by our group and binds with low nanomolar affinity to FIX, FIXa and FX, with no detectable binding to FXa. In one-stage clotting, BS-027125 achieved clot times similar to physiological levels of FVIII, but had poor activity in thrombin generation at these concentrations. Furthermore, it too maintained small amounts of phospholipid-independent activity in chromogenic FXa generation. Given the artificial nature of the chromogenic FXa generation assay, and that activity of prothrombinase is PS-dependent thereby precluding omission of phospholipids from thrombin generation assays, we developed an assay to detect FXa generation in a plasma milieu by FVIIIa mimetic antibodies or FVIII with and without phospholipid vesicles.Methods: FVIIIa mimetic antibodies or recombinant FVIII (rFVIII) were incubated with thrombin for 5 minutes, quenched with hirudin, then spiked into platelet-free congenital hemophilia A plasma treated with additional hirudin. FXIa (to generate FIXa in situ) with and without PC:PE:PS (40:40:20 molar ratio) phospholipid vesicles was added and reactions were triggered with a solution of CaCl2 and fluorogenic FXa substrate (Mes-D-LGR-ANSN(C2H5)2). Substrate cleavage was monitored kinetically on a fluorescent plate reader. Substrate cleavage by FXIa could not be detected, yet another unknown plasma peptidase did cleave substrate at a constant low rate that was background subtracted.Results: In the absence of phospholipid vesicles, SI-Emi maintained 51±3.7% of its FXa generation activity at all concentrations tested (3.8±0.4 versus 8.0±1.1 RFU/min at 333 nM). BS-027125 showed very low activity (0.43±0.12 RFU/min at 50 nM) in the presence of phospholipid vesicles, however, in the absence of phospholipid vesicles, BS-027125 activity was not detectable above baseline. Nearly all rFVIII activity (>99%) was lost in the absence of phospholipid vesicles (0.14±0.04 versus 15.1±1.8 RFU/min at 0.3 IU/mL). Addition of annexin V was sufficient to block all rFVIII activity in the presence or absence of phospholipid vesicles, but could not block SI-Emi activity. Furthermore, addition of rivaroxaban, a direct FXa inhibitor, confirmed that detection of substrate cleavage was due to FXa activity.Conclusions: In the absence of phosphatidylserine-containing phospholipid vesicles, SI-Emi promoted the generation of FXa in plasma triggered with FXIa. The activity of BS-027125 was too low in this assay to clearly determine its phospholipid-independent activity. These results suggest SI-Emi has mis-regulated (PS-independent) procoagulant activity due to a lack of phospholipid localization of the antibody-FIXa-FX complex. Given the weak affinity of SI-Emi for its antigens, the exact mechanism enabling this activity is unclear. Further study of this phenomenon and its relevance to overall thrombin generation and in vivo activity are needed. DisclosuresAleman:Bioverativ, a Sanofi company: Employment. Jindal:Bioverativ, a Sanofi company: Employment. Leksa:Bioverativ a Sanofi company: Employment. Peters:Bioverativ a Sanofi company: Employment, Equity Ownership. Salas:Bioverativ a Sanofi company: Employment, Equity Ownership.

REDOR NMR Reveals Multiple Conformers for a Protein Kinase C Ligand in a Membrane Environment.

Bryostatin 1 (henceforth bryostatin) is in clinical trials for the treatment of Alzheimer’s disease and for HIV/AIDS eradication. It is also a preclinical lead for cancer immunotherapy and other therapeutic indications. Yet nothing is known about the conformation of bryostatin bound to its protein kinase C (PKC) target in a membrane microenvironment. As a result, efforts to design more efficacious, better tolerated, or more synthetically accessible ligands have been limited to structures that do not include PKC or membrane effects known to influence PKC–ligand binding. This problem extends more generally to many membrane-associated proteins in the human proteome. Here, we use rotational-echo double-resonance (REDOR) solid-state NMR to determine the conformations of PKC modulators bound to the PKCδ-C1b domain in the presence of phospholipid vesicles. The conformationally limited PKC modulator phorbol diacetate (PDAc) is used as an initial test substrate. While unanticipated partitioning of PDAc between an immobilized protein-bound state and a mobile state in the phospholipid assembly was observed, a single conformation in the bound state was identified. In striking contrast, a bryostatin analogue (bryolog) was found to exist exclusively in a protein-bound state, but adopts a distribution of conformations as defined by three independent distance measurements. The detection of multiple PKCδ-C1b-bound bryolog conformers in a functionally relevant phospholipid complex reveals the inherent dynamic nature of cellular systems that is not captured with single-conformation static structures. These results indicate that binding, selectivity, and function of PKC modulators, as well as the design of new modulators, are best addressed using a dynamic multistate model, an analysis potentially applicable to other membrane-associated proteins.

The binding of activated Gαq to phospholipase C-β exhibits anomalous affinity

Upon activation by the Gq family of Gα subunits, Gβγ subunits, and some Rho family GTPases, phospholipase C-β (PLC-β) isoforms hydrolyze phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol. PLC-β isoforms also function as GTPase-activating proteins, potentiating Gq deactivation. To elucidate the mechanism of this mutual regulation, we measured the thermodynamics and kinetics of PLC-β3 binding to Gαq FRET and fluorescence correlation spectroscopy, two physically distinct methods, both yielded Kd values of about 200 nm for PLC-β3-Gαq binding. This Kd is 50-100 times greater than the EC50 for Gαq-mediated PLC-β3 activation and for the Gαq GTPase-activating protein activity of PLC-β. The measured Kd was not altered either by the presence of phospholipid vesicles, phosphatidylinositol 4,5-bisphosphate and Ca2+, or by the identity of the fluorescent labels. FRET-based kinetic measurements were also consistent with a Kd of 200 nm We determined that PLC-β3 hysteresis, whereby PLC-β3 remains active for some time following either Gαq-PLC-β3 dissociation or PLC-β3-potentiated Gαq deactivation, is not sufficient to explain the observed discrepancy between EC50 and Kd These results indicate that the mechanism by which Gαq and PLC-β3 mutually regulate each other is far more complex than a simple, two-state allosteric model and instead is probably kinetically determined.

One pathogen two stones: are Australian tree frog antimicrobial peptides synergistic against human pathogens?

Antimicrobial peptides (AMPs) may act by targeting the lipid membranes and disrupting the bilayer structure. In this study, three AMPs from the skin of Australian tree frogs, aurein 1.2, maculatin 1.1 and caerin 1.1, were investigated against Gram-negative Escherichia coli, Gram-positive Staphylococcus aureus, and vesicles that mimic their lipid compositions. Furthermore, equimolar mixtures of the peptides were tested to identify any synergistic interactions in antimicrobial activity. Minimum inhibition concentration and minimum bactericidal concentration assays showed significant activity against S. aureus but not against E. coli. Aurein was the least active while maculatin was the most active peptide and some synergistic effects were observed against S. aureus. Circular dichroism experiments showed that, in the presence of phospholipid vesicles, the peptides transitioned from an unstructured to a predominantly helical conformation (>50%), with greater helicity for POPG/TOCL compared to POPE/POPG vesicles. The helical content, however, was less in the presence of live E. coli and S. aureus, 25 and 5%, respectively. Equimolar concentrations of the peptides did not appear to form greater supramolecular structures. Dye release assays showed that aurein required greater concentration than caerin and maculatin to disrupt the lipid bilayers, and mixtures of the peptides did not cooperate to enhance their lytic activity. Overall, aurein, maculatin, and caerin showed moderate synergy in antimicrobial activity against S. aureus without becoming more structured or enhancement of their membrane-disrupting activity in phospholipid vesicles.

Effects of impaired membrane interactions on α-synuclein aggregation and neurotoxicity

The post-mortem brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein's aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein's neurotoxicity in PD and other synucleinopathy disorders.

Interactions of amelogenin with phospholipids.

Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.

Spectroscopic Study on the Interaction of 4-dimethylaminochalcones with Phospholipids

The ultraviolet-visible and fluorescence spectroscopic properties of 4′-dimethylaminochalcone (1a) and its cyclic analogs 2a–4a have been studied in the presence of phospholipid vesicles (i.e., egg yolk lecithin and dipalmitoylpho sphatidylcholine), bovine serum albumin (BSA), and lipoprotein particles (i.e., bovine serum albumin plus egg yolk lecithin). The spectral results showed that compounds 1a–4a formed hydrophobic interactions with the phospholipids, lipoproteins, and BSA at the polar/nonpolar interface. Compounds 3a and 4a exhibited the strongest hydrophobic interactions of all of the compounds tested towards the phospholipids. Compound 2a gave the best fluorescent fluorophore indicating interactions with the lipids, lipoproteins, and proteins. Fluorescent microscopic imaging of breast cancer cells treated with compounds 1a–4a revealed that they could be used to stain all of the cellular components and destroy the nuclear structure. Compounds 1a–4a were found to be concentrated predominantly on the surfaces of the liposomes and lipoproteins.

GP41 Ectodomain Dissociates and Forms a Stable Monomer on Phospholipid Vesicles and Detergent Micelles: Implication for the HIV-1 Env-Mediated Membrane Fusion

The first step of HIV infection involves the fusion of the viral and target cell membranes, a process mediated by the viral envelope glycoproteins, gp120 and gp41. The binding of gp120 to the cell surface receptors CD4 triggers a cascade of conformational changes that disrupt the gp41-gp120 interactions and allows the insertion of the N-terminal fusion peptide of gp41 into the host cell membrane. The gp41 trimer then rapidly folds into a 6-helix bundle that pulls together the fusion peptide, inserted in the host cell membrane and the transmembrane domain, located in the viral membrane. Very little structural information is known about the pre-fusion state of gp41 notwithstanding its critical importance for the design of fusion inhibitors. To investigate the dynamics and structural properties of such metastable states, we designed a set of protein constructs mimicking the extracellular ectodomain of gp41. We found that the secondary structure of gp41 ectodomain is pronouncedly impacted by the presence of phospholipid vesicles or detergents containing phosphatidyl choline head groups. Our NMR and multi-angle light scattering data attribute these changes to the transition between a trimer in the absence of detergent to a monomer in the presence of a membrane-mimicking environment. The structure of gp41 in a monomeric state was determined by state-of-the-art NMR techniques, including measurements of NOE distance restraints and residual dipolar couplings in weakly aligned solutions. 15N relaxation data provided a detailed view at the backbone dynamics of both the trimeric and monomeric conformations. This stable monomeric state may represent a crucial structural intermediate facilitating both the gp41 conformational change during fusion and the local apposition of the viral and cellular membranes.

Structural Intermediates during α-Synuclein Fibrillogenesis on Phospholipid Vesicles

α-Synuclein (AS) fibrils are the main protein component of Lewy bodies, the pathological hallmark of Parkinson's disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to β-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to β-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the N-terminal domain with a partial disruption of the long β-strand located in the 40s and small perturbations in residues located in the "non-β amyloid component" (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles.

WT alpha-synuclein fibrils in the presence of phospholipid vesicles

WT alpha-synuclein fibrils in the presence of phospholipid vesicles

Partitioning of 2,6-Bis(1 H-Benzimidazol-2-yl)pyridine fluorophore into a phospholipid bilayer: Complementary use of fluorescence quenching studies and molecular dynamics simulations

Successful use of fluorescence sensing in elucidating the biophysical properties of lipid membranes requires knowledge of the distribution and location of an emitting molecule in the bilayer. We report here that 2,6-bis(1 H-benzimidazol-2-yl)pyridine (BBP), which is almost non-fluorescent in aqueous solutions, reveals a strong emission enhancement in a hydrophobic environment of a phospholipid bilayer, making it interesting for fluorescence probing of water content in a lipid membrane. Comparing the fluorescence behavior of BBP in a wide variety of solvents with those in phospholipid vesicles, we suggest that the hydrogen bonding interactions between a BBP fluorophore and water molecules play a crucial role in the observed “light switch effect”. Therefore, the loss of water-induced fluorescence quenching inside a membrane are thought to be due to deep penetration of BBP into the hydrophobic, water-free region of a bilayer. Characterized by strong quenching by transition metal ions in solution, BBP also demonstrated significant shielding from the action of the quencher in the presence of phospholipid vesicles. We used the increase in fluorescence intensity, measured upon titration of probe molecules with lipid vesicles, to estimate the partition constant and the Gibbs free energy (ΔG) of transfer of BBP from aqueous buffer into a membrane. Partitioning BBP revealed strongly favorable ΔG, which depends only slightly on the lipid composition of a bilayer, varying in a range from − 6.5 to − 7.0 kcal/mol. To elucidate the binding interactions of the probe with a membrane on the molecular level, a distribution and favorable location of BBP in a POPC bilayer were modeled via atomistic molecular dynamics (MD) simulations using two different approaches: (i) free, diffusion-driven partitioning of the probe molecules into a bilayer and (ii) constrained umbrella sampling of a penetration profile of the dye molecule across a bilayer. Both of these MD approaches agreed with regard to the preferred location of a BBP fluorophore within the interfacial region of a bilayer, located between the hydrocarbon acyl tails and the initial portion of the lipid headgroups. MD simulations also revealed restricted permeability of water molecules into this region of a POPC bilayer, determining the strong fluorescence enhancement observed experimentally for the membrane-partitioned form of BBP.

Rheological study of hydrophobically modified hydroxyethyl cellulose and phospholipid vesicles

The rheological behavior of hydrophobically modified hydroxyehtyl cellulose (HMHEC) was studied in the presence of phospholipid vesicles. The effects of sonication, lipid species and concentration on the rheology were investigated at two HMHEC concentrations: 0.3 wt.% and 0.7 wt.%. It is found that compared to a pure HMHEC solution, the mixture viscosity could be enhanced by more than one order of magnitude for sonicated samples at certain lipid concentrations. The viscosity, which depends on the vesicle size and concentration, first increases with the lipid concentration and then decreases. Besides, the lipid addition increases the plateau modulus. These observed rheological behaviors can be explained by two types of association; some of the hydrophobes aggregate to form micelles, while others are embedded in the vesicle bilayers. By this means, the vesicles can be interconnected, contributing to the viscosity enhancement.

Reaction Mechanism of Superoxide Generation during Ubiquinol Oxidation by the Cytochrome bc1 Complex

In addition to its main functions of electron transfer and proton translocation, the cytochrome bc(1) complex (bc(1)) also catalyzes superoxide anion (O(2)(*)) generation upon oxidation of ubiquinol in the presence of molecular oxygen. The reaction mechanism of superoxide generation by bc(1) remains elusive. The maximum O(2)(*) generation activity is observed when the complex is inhibited by antimycin A or inactivated by heat treatment or proteinase K digestion. The fact that the cytochrome bc(1) complex with less structural integrity has higher O(2)(*)-generating activity encouraged us to speculate that O(2)(*) is generated inside the complex, perhaps in the hydrophobic environment of the Q(P) pocket through bifurcated oxidation of ubiquinol by transferring its two electrons to a high potential electron acceptor, iron-sulfur cluster, and a low potential heme b(L) or molecular oxygen. If this speculation is correct, then one should see more O(2)(*) generation upon oxidation of ubiquinol by a high potential oxidant, such as cytochrome c or ferricyanide, in the presence of phospholipid vesicles or detergent micelles than in the hydrophilic conditions, and this is indeed the case. The protein subunits, at least those surrounding the Q(P) pocket, may play a role either in preventing the release of O(2)(*) from its production site to aqueous environments or in preventing O(2) from getting access to the hydrophobic Q(P) pocket and might not directly participate in superoxide production.

Cleavage at Arg-1689 Influences Heavy Chain Cleavages during Thrombin-catalyzed Activation of Factor VIII

The procofactor, factor VIII, is activated by thrombin or factor Xa-catalyzed cleavage at three P1 residues: Arg-372, Arg-740, and Arg-1689. The catalytic efficiency for thrombin cleavage at Arg-740 is greater than at either Arg-1689 or Arg-372 and influences reaction rates at these sites. Because cleavage at Arg-372 appears rate-limiting and dependent upon initial cleavage at Arg-740, we investigated whether cleavage at Arg-1689 influences catalysis at this step. Recombinant B-domainless factor VIII mutants, R1689H and R1689Q were prepared and stably expressed to slow and eliminate cleavage, respectively. Specific activity values for the His and Gln mutations were approximately 50 and approximately 10%, respectively, that of wild type. Thrombin activation of the R1689H variant showed an approximately 340-fold reduction in the rate of Arg-1689 cleavage, whereas the R1689Q variant was resistant to thrombin cleavage at this site. Examination of heavy chain cleavages showed approximately 4- and 11-fold reductions in A2 subunit generation and approximately 3- and 7-fold reductions in A1 subunit generation for the R1689H and R1689Q mutants, respectively. These results suggest a linkage between light chain cleavage and cleavages in heavy chain. Results obtained evaluating proteolysis of the factor VIII mutants by factor Xa revealed modest rate reductions (<5-fold) in generating A2 and A1 subunits and in cleaving light chain at Arg-1721 from either variant, suggesting little dependence upon prior cleavage at residue 1689 as compared with thrombin. Overall, these results are consistent with a competition between heavy and light chains for thrombin exosite binding and subsequent proteolysis with binding of the former chain preferred.

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin.