Enhanced Membrane Affinity Research Articles

Force Mapping Study of Actinoporin Effect in Membranes Presenting Phase Domains.

Equinatoxin II (EqtII) and Fragaceatoxin C (FraC) are pore-forming toxins (PFTs) from the actinoporin family that have enhanced membrane affinity in the presence of sphingomyelin (SM) and phase coexistence in the membrane. However, little is known about the effect of these proteins on the nanoscopic properties of membrane domains. Here, we used combined confocal microscopy and force mapping by atomic force microscopy to study the effect of EqtII and FraC on the organization of phase-separated phosphatidylcholine/SM/cholesterol membranes. To this aim, we developed a fast, high-throughput processing tool to correlate structural and nano-mechanical information from force mapping. We found that both proteins changed the lipid domain shape. Strikingly, they induced a reduction in the domain area and circularity, suggesting a decrease in the line tension due to a lipid phase height mismatch, which correlated with proteins binding to the domain interfaces. Moreover, force mapping suggested that the proteins affected the mechanical properties at the edge, but not in the bulk, of the domains. This effect could not be revealed by ensemble force spectroscopy measurements supporting the suitability of force mapping to study local membrane topographical and mechanical alterations by membranotropic proteins.

Chemical Interactions of Cryptic Actinomycete Metabolite 5‐Alkyl‐1,2,3,4‐tetrahydroquinolines through Aggregate Formation

AbstractOrganisms often produce secondary metabolites as a mixture of biosynthetically related congeners. However, why are metabolites with minor chemical variations produced simultaneously? 5‐Alkyl‐1,2,3,4‐tetrahydroquinolines (5aTHQs) are small, lipophilic metabolites produced by Streptomyces nigrescens HEK616 when cultured with Tsukamurella pulmonis TP‐B0596. A mixture of 5aTHQs forms aggregates that show enhanced membrane affinity and biological activity. The ability to form aggregates and membrane‐binding activity is regulated by the length of the alkyl chains. Aggregates with long alkyl chains were too stable to fuse with lipid membranes. However, if inactive 5aTHQ congener was mixed with active congener, the mixture showed increased membrane affinity, enabling cellular entry and biological activity. Therefore, it is shown that sloppiness in a biosynthetic pathway, by which minor structural variations can be produced, is functionally rational, as the metabolites show synergistic action.

Chemical Interactions of Cryptic Actinomycete Metabolite 5-Alkyl-1,2,3,4-tetrahydroquinolines through Aggregate Formation.

Organisms often produce secondary metabolites as a mixture of biosynthetically related congeners. However, why are metabolites with minor chemical variations produced simultaneously? 5-Alkyl-1,2,3,4-tetrahydroquinolines (5aTHQs) are small, lipophilic metabolites produced by Streptomyces nigrescens HEK616 when cultured with Tsukamurella pulmonis TP-B0596. A mixture of 5aTHQs forms aggregates that show enhanced membrane affinity and biological activity. The ability to form aggregates and membrane-binding activity is regulated by the length of the alkyl chains. Aggregates with long alkyl chains were too stable to fuse with lipid membranes. However, if inactive 5aTHQ congener was mixed with active congener, the mixture showed increased membrane affinity, enabling cellular entry and biological activity. Therefore, it is shown that sloppiness in a biosynthetic pathway, by which minor structural variations can be produced, is functionally rational, as the metabolites show synergistic action.

Membrane affinity and fluorescent labelling: comparative study of monolayer interaction, cellular uptake and cytotoxicity profile of carboxyfluorescein-conjugated cationic peptides.

Fluorescent labelling is a common approach to reveal the molecular details of cellular uptake, internalisation, transport, distribution processes in biological systems. The conjugation with a fluorescent moiety might affect relevant physico-chemical and in vitro transport properties of the bioactive component. A representative set of seven cationic peptides-including cell-penetrating peptides as well as antimicrobial peptides and synthetic derivatives-was selected for our comparative study. Membrane affinity of the peptides and their 5(6)-carboxyfluorescein (Cf) derivatives was determined quantitatively and compared applying Langmuir monolayer of zwitterionic (DPPC) and negatively charged (DPPC + DPPG) lipids as cell membrane models. The interaction with neutral lipid layer is mainly governed by the overall hydrophobicity of the molecule which is remarkably increased by Cf-conjugation for the most hydrophobic Magainin, Melittin and Transportan. A significantly enhanced membrane affinity was detected in negatively charged lipid model monolayer for all of the peptides since the combination of electrostatic and hydrophobic interaction is active in that case. The Cf-conjugation improved the penetration ability of Penetratin and Dhvar4 suggesting that both the highly charged character (Z/n) and the increased hydrophobicity by Cf-conjugation present important contribution to membrane interaction. This effect might also responsible for the observed high in vitro internalisation rate of Penetratin and Dhvar4, while according to in vitro studies they did not cause damage of cell membrane. From the experiments with the given seven cationic peptides, it can be concluded that the Cf-conjugation alters the degree of membrane interaction of such peptides which are moderately hydrophobic and highly charged.

Aggregation-induced conformation changes dictate islet amyloid polypeptide (IAPP) membrane affinity

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates (“oligomers”) of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic “oligomer fold” may not be universal.

Protein-inspired antibiotics active against vancomycin- and daptomycin-resistant bacteria

The public health threat posed by a looming ‘post-antibiotic’ era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature’s control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the ‘vancapticins’, possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to ‘revitalise’ antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.

Single-Molecule Studies Reveal a Hidden Key Step in the Activation Mechanism of Membrane-Bound Protein Kinase C-α

Protein kinase C-α (PKCα) is a member of the conventional family of protein kinase C isoforms (cPKCs) that regulate diverse cellular signaling pathways, share a common activation mechanism, and are linked to multiple pathologies. The cPKC domain structure is modular, consisting of an N-terminal pseudosubstrate peptide, two inhibitory domains (C1A and C1B), a targeting domain (C2), and a kinase domain. Mature, cytoplasmic cPKCs are inactive until they are switched on by a multistep activation reaction that occurs largely on the plasma membrane surface. Often, this activation begins with a cytoplasmic Ca2+ signal that triggers C2 domain targeting to the plasma membrane where it binds phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). Subsequently, the appearance of the signaling lipid diacylglycerol (DAG) activates the membrane-bound enzyme by recruiting the inhibitory pseudosubstrate and one or both C1 domains away from the kinase domain. To further investigate this mechanism, this study has utilized single-molecule total internal reflection fluorescence microscopy (TIRFM) to quantitate the binding and lateral diffusion of full-length PKCα and fragments missing specific domain(s) on supported lipid bilayers. Lipid binding events, and events during which additional protein is inserted into the bilayer, were detected by their effects on the equilibrium bound particle density and the two-dimensional diffusion rate. In addition to the previously proposed activation steps, the findings reveal a major, undescribed, kinase-inactive intermediate. On bilayers containing PS or PS and PIP2, full-length PKCα first docks to the membrane via its C2 domain, and then its C1A domain embeds itself in the bilayer even before DAG appears. The resulting pre-DAG intermediate with membrane-bound C1A and C2 domains is the predominant state of PKCα while it awaits the DAG signal. The newly detected, membrane-embedded C1A domain of this pre-DAG intermediate confers multiple useful features, including enhanced membrane affinity and longer bound state lifetime. The findings also identify the key molecular step in kinase activation: because C1A is already membrane-embedded in the kinase off state, recruitment of C1B to the bilayer by DAG or phorbol ester is the key regulatory event that stabilizes the kinase on state. More broadly, this study illustrates the power of single-molecule methods in elucidating the activation mechanisms and hidden regulatory states of membrane-bound signaling proteins.

Dissociation of the K-Ras4B/PDEδ Complex upon Contact with Lipid Membranes: Membrane Delivery Instead of Extraction

K-Ras4B is a small GTPase whose selective membrane localization and clustering into microdomains are mediated by its polybasic farnesylated C-terminus. The importance of the subcellular distribution for the signaling activity of K-Ras4B became apparent from recent in vivo studies, showing that the delta subunit of cGMP phosphodiesterase (PDEδ), which possesses a hydrophobic prenyl-binding pocket, is able to function as a potential binding partner for farnesylated proteins, thereby leading to a modulation of the spatiotemporal organization of K-Ras. Even though PDEδ has been suggested to serve as a cytosolic carrier for Ras, the functional transport mechanism still remains largely elusive. In this study, the effect of PDEδ on the interaction of GDP- and GTP-loaded K-Ras4B with neutral and anionic model biomembranes has been investigated by a combination of different spectroscopic and imaging techniques. The results show that PDEδ is not able to extract K-Ras4B from membranes. Rather, the K-Ras4B/PDEδ complex formed in bulk solution turned out to be unstable in the presence of heterogeneous membranes, resulting in a release of farnesylated K-Ras4B upon membrane contact. With the additional observation of enhanced membrane affinity for the K-Ras4B/PDEδ complex, a molecular mechanism for the PDEδ-K-Ras4B-membrane interaction could be proposed. This includes an effective delivery of PDEδ-solubilized K-Ras4B to the plasma membrane, probably through cytoplasmic diffusion, the dissociation of the K-Ras4B/PDEδ complex upon plasma membrane contact, and finally the membrane binding of released farnesylated K-Ras4B that leads to K-Ras4B-enriched microdomain formation.

Selective Acylation Enhances Membrane Charge Sensitivity of the Antimicrobial Peptide Mastoparan-X

The partitioning of the wasp venom peptide mastoparan-X (MPX) into neutral and negatively charged lipid membranes has been compared with two new synthetic analogs of MPX where the N α -terminal of MPX was acylated with propanoic acid (PA) and octanoic acid (OA). The acylation caused a considerable change in the membrane partitioning properties of MPX and it was found that the shorter acylation with PA gave improved affinity and selectivity toward negatively charged membranes, whereas OA decreased the selectivity. Based on these findings, we hypothesize that minor differences in the embedding and positioning of the peptide in the membrane caused by either PA or OA acylation play a critical role in the fine-tuning of the effective charge of the peptide and thereby the fine-tuning of the peptide's selectivity between neutral and negatively charged lipid membranes. This finding is unique compared to previous reports where peptide acylation enhanced membrane affinity but also resulted in impaired selectivity. Our result may provide a method of enhancing selectivity of antimicrobial peptides toward bacterial membranes due to their high negative charge—a finding that should be investigated for other, more potent antimicrobial peptides in future studies.

Lipidation by the Host Prenyltransferase Machinery Facilitates Membrane Localization of Legionella pneumophila Effector Proteins

The intracellular human pathogen Legionella pneumophila translocates multiple proteins in the host cytosol known as effectors, which subvert host cellular processes to create a membrane-bound organelle that supports bacterial replication. It was observed that several Legionella effectors encode a prototypical eukaryotic prenylation CAAX motif (where C represents a cysteine residue and A denotes an aliphatic amino acid). These bacterial motifs mediated posttranslational modification of effector proteins resulting in the addition of either a farnesyl or geranylgeranyl isoprenyl lipid moiety to the cysteine residue of the CAAX tetrapeptide. Lipidation enhanced membrane affinity for most Legionella CAAX motif proteins and facilitated the localization of these effector proteins to host organelles. Host farnesyltransferase and class I geranylgeranyltransferase were both involved in the lipidation of the Legionella CAAX motif proteins. Perturbation of the host prenylation machinery during infection adversely affected the remodeling of the Legionella-containing vacuole. Thus, these data indicate that Legionella utilize the host prenylation machinery to facilitate targeting of effector proteins to membrane-bound organelles during intracellular infection.

Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma

Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca(2+) titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca(2+) requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity.

Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain

The regulation of PTEN intrinsic biochemical properties has not been fully elucidated. In this report, we investigated the role of the PTEN carboxyl-terminal tail domain in regulating its membrane targeting and catalytic functions. Characterization of a panel of PTEN phosphorylation site mutants revealed that mutating Ser-385 to alanine (S385A) promoted membrane localization in vivo and phosphatase activity in vitro. Furthermore, S385A mutation was associated with a substantial reduction in the phosphorylation of the Ser-380/Thr-382/Thr-383 cluster. Therefore, Ser-385 could prime additional dephosphorylation events to regulate PTEN catalytic activity. Moreover, substituting Ser-380/Thr-382/Thr-383 to phosphomimic residues reversed the phosphatase activity of the S385A mutation. Next, we further defined the underlying mechanisms responsible for the COOH-terminal tail region in modulating PTEN biological activity. We have identified an interaction between the 71-amino acid carboxyl-terminal tail region and the CBRIII motif of the C2 domain, which has been implicated in membrane binding. In addition, a synthetic phosphomimic peptide encompassing the phosphorylation site cluster between amino acids 368 and 390 within the tail region mediated the suppression of PTEN catalytic activity in vitro. This same peptide when expressed in cultured cells also impeded PTEN membrane localization and enhanced phospho-Akt levels. Thus, our data suggest that the COOH-terminal tail can act as an autoinhibitory domain to control both PTEN membrane recruitment and phosphatase activity.

Improved Cell Viability of Linear Polyethylenimine through γ‐Cyclodextrin Inclusion for Effective Gene Delivery

A polypseudorotaxane consisting of a linear polyethylenimine with Mn of 22,000 (LPEI22k) and gamma-cyclodextrins (gamma-CDs; LPEI22k/gamma-CD) has been examined as a gene carrier. The polyplex formation with luciferase-encoding plasmid DNA (pDNA), intracellular trafficking of polyplex, cytotoxicity, and transfection efficiency were evaluated by various characteristic methods. LPEI22k/gamma-CD formed a pDNA polyplex at higher N/P ratios than LPEI22k; this suggests that the gamma-CD threading sterically interfered with the polyplex formation. In addition, the zeta potentials of the polyplex significantly decreased due to the reduction in charge density of LPEI22k caused by gamma-CD threading. The cellular uptake of pDNA in the LPEI22k/gamma-CD polyplex was enhanced by free gamma-CDs released from the polyplex that might accelerate the cellular uptake through enhanced membrane affinity. LPEI22k/gamma-CD significantly increased cell viability even at high N/P ratios, and the polyplex showed high transfection efficacy. The low cytotoxicity and high gene expression of LPEI22k/gamma-CD are advantageous to polyplex administration in vivo.

Proapoptotic BH3-only proteins trigger membrane integration of prosurvival Bcl-w and neutralize its activity.

Prosurvival Bcl-2–like proteins, like Bcl-w, are thought to function on organelles such as the mitochondrion and to be targeted to them by their hydrophobic COOH-terminal domain. We unexpectedly found, however, that the membrane association of Bcl-w was enhanced during apoptosis. In healthy cells, Bcl-w was loosely attached to the mitochondrial membrane, but it was converted into an integral membrane protein by cytotoxic signals that induce binding of BH3-only proteins, such as Bim, or by the addition of BH3 peptides to lysates. As the structure of Bcl-w has revealed that its COOH-terminal domain occupies the hydrophobic groove where BH3 ligands bind, displacement of that domain by a BH3 ligand would displace the hydrophobic COOH-terminal residues, allowing their insertion into the membrane. To determine whether BH3 ligation is sufficient to induce the enhanced membrane affinity, or to render Bcl-w proapoptotic, we mimicked their complex by tethering the Bim BH3 domain to the NH2 terminus of Bcl-w. The chimera indeed bound avidly to membranes, in a fashion requiring the COOH-terminal domain, but neither promoted nor inhibited apoptosis. These results suggest that ligation of a proapoptotic BH3-only protein alters the conformation of Bcl-w, enhances membrane association, and neutralizes its survival function.

Response of Factor VIII and IX-Deficient Blood to Wild Type and High Membrane Affinity Mutant Factor VIIa in an In Vitro Whole Blood Clotting Assay: Possible Correlation to Clinical Outcome

A modified form of the activated whole blood clotting time was used to evaluate response of blood from hemophilia patients to factor VIIa. Repeated assays of individuals over a one-year period showed consistency for each individual and significant difference between individuals. Four hemophilia patients with inhibitors gave low response to factor VIIa in the assay and were characterized as low or moderate clinical responders to factor VIIa therapy. Another four patients with moderate to good clinical responsiveness to factor VIIa therapy showed a strong response to factor VIIa in the assay. A factor VIIa mutant with enhanced membrane affinity showed 10- to 13-fold higher activity than wild type factor VIIa. The results may justify larger studies to determine the utility of this assay for evaluation of patient responsiveness and to set factor VIIa therapy levels.

Assembly and Release of Human Immunodeficiency Virus Type 1 Gag Proteins Containing Tandem Repeats of the Matrix Protein Coding Sequences in the Matrix Domain

We have constructed human immunodeficiency virus (HIV) gag mutants by increasing the matrix protein (MA) sequences via tandemly repeated duplication of the central 107-MA codons. Instead of a total of 132 amino acid residues for the wild-type MA, the resultant mutants designated as MA2, MA3, and MA4 contained a total of 242, 352, and 462 codons in the MA domains, respectively. Analysis indicated that the addition of 110 or 220 amino acid residues to the MA did not significantly affect the assembly, release, and processing of particles; however, particle production was markedly reduced when another copy of 110 residues was added to the MA. Subcellular fractionation analysis suggested that the MA tandem repeat mutations enhanced the Gag membrane affinity, in a manner which correlated with the copy number of MA sequences. The effects of enhanced membrane affinity were substantially reduced when sequences downstream of the capsid (CA) domain were deleted. Sucrose density gradient fractionation analysis showed that particles produced by the large insertion mutants possessed wild-type (wt) HIV particle density. Truncation of sequences downstream of the nucleocapsid (NC) domains of the mutants did not influence the budding of particles. In contrast, particle budding was severely impaired when sequences downstream of the CA domain were truncated. Particle densities for the large Gag proteins, which were truncated at the C-terminus of CA, were about 1.12–1.14 g/ml lower than that for wt. Our results suggest that the HIV MA domain could adopt insertions of large protein sequences, and strongly support the proposal that the NC and p2 domains play a crucial role in the process of correct Gag protein packing.