Spatial Configuration and Composition of Charge Modulates Transport into a Mucin Hydrogel Barrier

The mucus barrier is selectively permeable to a wide variety of molecules, proteins, and cells, and establishes gradients of these particulates to influence the uptake of nutrients, the defense against pathogens, and the delivery of drugs. Despite its importance for health and disease, the criteria that govern transport through the mucus barrier are largely unknown. Studies with uniformly functionalized nanoparticles have provided critical information about the relevance of particle size and net charge for mucus transport. However, these particles lack the detailed spatial arrangements of charge found in natural mucus-interacting substrates, such as certain viruses, which may have important consequences for transport through the mucus barrier. Using a novel, to our knowledge, microfluidic design that enables us to measure real-time transport gradients inside a hydrogel of mucins, the gel-forming glycoprotein component of mucus, we show that two peptides with the same net charge, but different charge arrangements, exhibit fundamentally different transport behaviors. Specifically, we show that certain configurations of positive and negative charges result in enhanced uptake into a mucin barrier, a remarkable effect that is not observed with either charge alone. Moreover, we show that the ionic strength within the mucin barrier strongly influences transport specificity, and that this effect depends on the detailed spatial arrangement of charge. These findings suggest that spatial charge distribution is a critical parameter to modulate transport through mucin-based barriers, and have concrete implications for the prediction of mucosal passage, and the design of drug delivery vehicles with tunable transport properties.     

The engineering of membrane-permeable peptides

Reversible lipid attachment was investigated as a means to deliver small peptides into cells. Two labile straight chain alkyl motifs were developed: a cysteine dodecane disulfide (Cdd) building block and a tyrosine- or serine-myristate ester. Both moieties are cleaved on cell internalization and are compatible with Fmoc solid phase peptide synthesis. A series of fluorophore-labeled peptides that varied in lipophilic content, net charge, and charge distribution were synthesized. The peptides were screened for cellular uptake efficiency as monitored by fluorescence microscopy. Effective peptide transport is based on a distributed net positive charge introduced as lysine residues at the C and/or N terminus of the peptide and the presence of a hydrophobic domain exhibiting an estimated log P4.0. The incorporation of labile lipid motifs into peptides enhances lipophilic character of the peptides and contributes to cellular uptake with minimal alteration to the native sequence.     

Kinetics of nanochain formation in a simplified model of amelogenin biomacromolecules

We show that the kinetics of nanochain formation of amelogenin molecules is well described by a combination of translational and rotational diffusion of a simplified anisotropic bipolar model consisting of hydrophobic spherical colloid particles and a point charge located on each particle surface. The colloid particles interact via a standard depletion attraction whereas the point charges interact through a screened Coulomb repulsion. We study the kinetics via a Brownian dynamics simulation of both translational and rotational motions and show that the anisotropy brought in by the charge dramatically affects the kinetic pathway of cluster formation and our simple model captures the main features of the experimental observations.     

The role of the N region in signal sequence and signal-anchor function.

To determine the role of sequences other than the hydrophobic core in mediating signal sequence function, we examined the behavior of fusion proteins and deletion mutants in cell-free systems. We demonstrate that neither the N nor the C region of the preprolactin signal sequence is necessary for translocation. However, insertion of sequences with either a net charge of +2.5 or -6.0 between the N region and the hydrophobic core of the signal converted it into a signal-anchor. The topologies adopted (types I and II, respectively) were opposite those predicted from the distribution of charges surrounding the hydrophobic core of the signals. When these mutant signals were located in the interior of an otherwise secreted protein, both sequences functioned as stop-transfer sequences. Related mutations were assayed in fusion proteins in which the IgM transmembrane domain functioned as an amino-terminal signal-anchor. For these molecules, the distribution of charged residues surrounding the hydrophobic core had no influence on the topology adopted. Our results suggest that features other than simple charge distribution play an important role in determining membrane topology in vitro.     

Interaction of 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes

We investigated the interaction of six 18-residue peptides derived from amphipathic helical segments of globular proteins with model membranes. The net charge of the peptides at neutral pH varies from −1 to +6. Circular dichroism spectra indicate that peptides with a high net positive charge tend to fold into a helical conformation in the presence of negatively charged lipid vesicles. In helical conformation, their average hydrophobic moment and hydrophobicity would render them surface-active. The composition of amino acids on the polar face of the helix in the peptides is considerably different. The peptides show variations in their ability to permeabilise zwitterionic and anionic lipid vesicles. Whereas increased net positive charge favours greater permeabilisation, the distribution of charged residues in the polar face also plays a role in determining membrane activity. The distribution of amino acids in the polar face of the helix in the peptides that were investigated do not fall into the canonical classes described. Amphipathic helices, which are part of proteins, with a pattern of amino acid distribution different from those observed in class L, A and others, could help in providing newer insights into peptide-membrane interactions.     

Signal peptides: exquisitely designed transport promoters

Prokaryotic proteins destined for transport out of the cytoplasm typically contain an N-terminal extension sequence, called the signal peptide, which is required for export, it is evident that many secretory proteins utilize a common export system, yet the signal sequences themselves display very little primary sequence homology. in attempting to understand how different signal peptides are able to promote protein secretion through the same pathway, the physical features of natural signal sequences have been extensively examined for similarities that might play a part in function. Experimental data have confirmed statistical analyses which highlighted dominant features of natural signal sequences in Escherichia coli: a net positive charge in the N-terminus increases efficiency of transport; the core region must maintain a threshold level of hydrophoblcity within a range of length limitations; the central portion adopts an α-hellcal conformation in hydrophobic environments; and the signal cleavage region is ideally six residues long, with small side-chain amino acids in the −1 and −3 positions. This review focuses on the parallels between signal peptide physical features and their functions, which emerge when the results of a variety of experimental approaches are combined. The requirement for each property may be ascribed to a potential interaction that is critical for efficient protein export. The summation of the key physical features produces signal peptides with the flexibility to function in multiple roles in order to expedite secretion. In this way, nature has indeed evolved exquisitely tuned signal sequences.     

Peptide specificity and lipid activation of the lysosomal transport complex ABCB9 (TAPL)

The lysosomal ABC transporter associated with antigen processing-like (TAPL, ABCB9) acts as an ATP-dependent polypeptide transporter with broad length selectivity. To characterize in detail its substrate specificity, a procedure for functional reconstitution of human TAPL was developed. By intensive screening of detergents, ideal solubilization conditions were evolved with respect to efficiency, long term stability, and functionality of TAPL. TAPL was isolated in a two-step procedure with high purity and, subsequently, reconstituted into proteoliposomes. The peptide transport activity of reconstituted TAPL strongly depends on the lipid composition. With the help of combinatorial peptide libraries, the key positions of the peptides were localized to the N- and C-terminal residues with respect to peptide transport. At both ends, TAPL favors positively charged, aromatic, or hydrophobic residues and disfavors negatively charged residues as well as asparagine and methionine. Besides specific interactions of both terminal residues, electrostatic interactions are important, since peptides with positive net charge are more efficiently transported than negatively charged ones.     

Structural requirements for cellular uptake of α‐helical amphipathic peptides

The structure of the cell‐permeable α‐helical amphipathic model peptide FLUOS‐KLALKLALKALKAALKLA‐NH₂ ( I ) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 μm . These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non‐amphipathic 18‐mer peptides with different primary structure, net charge and helix parameters from I . The amphipathic counterparts were internalized into the cells to a comparable extent as I , whereas no cellular uptake could be detected for the non‐amphipathic analogues. The mode of uptake remains unclear and involves both temperature‐sensitive and ‐insensitive processes, indicating non‐endocytic contributions. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of Particle Translocation through Mucin Hydrogels

Biological functional entities surround themselves with selective barriers that control the passage of certain classes of macromolecules while rejecting others. A prominent example of such a selective permeability barrier is given by mucus. Mucus is a biopolymer-based hydrogel that lines all wet epithelial surfaces of the human body. It regulates the uptake of nutrients from our gastrointestinal system, adjusts itself with the menstrual cycle to control the passage of sperm, and shields the underlying cells from pathogens such as bacteria and viruses. In the case of drug delivery, the mucus barrier needs to be overcome for successful medical treatment. Despite its importance for both physiology and medical applications, the underlying principles which regulate the permeability of mucus remain enigmatic. Here, we analyze the mobility of microscopic particles in reconstituted mucin hydrogels. We show that electrostatic interactions between diffusing particles and mucin polymers regulate the permeability properties of reconstituted mucin hydrogels. As a consequence, various parameters such as particle surface charge and mucin density, and buffer conditions such as pH and ionic strength, can modulate the microscopic barrier function of the mucin hydrogel. Our findings suggest that the permeability of a biopolymer-based hydrogel such as native mucus can be tuned to a wide range of settings in different compartments of our bodies.     

Lipid-packing perturbation of model membranes by pH-responsive antimicrobial peptides

The indiscriminate use of conventional antibiotics is leading to an increase in the number of resistant bacterial strains, motivating the search for new compounds to overcome this challenging problem. Antimicrobial peptides, acting only in the lipid phase of membranes without requiring specific membrane receptors as do conventional antibiotics, have shown great potential as possible substituents of these drugs. These peptides are in general rich in basic and hydrophobic residues forming an amphipathic structure when in contact with membranes. The outer leaflet of the prokaryotic cell membrane is rich in anionic lipids, while the surface of the eukaryotic cell is zwitterionic. Due to their positive net charge, many of these peptides are selective to the prokaryotic membrane. Notwithstanding this preference for anionic membranes, some of them can also act on neutral ones, hampering their therapeutic use. In addition to the electrostatic interaction driving peptide adsorption by the membrane, the ability of the peptide to perturb lipid packing is of paramount importance in their capacity to induce cell lysis, which is strongly dependent on electrostatic and hydrophobic interactions. In the present research, we revised the adsorption of antimicrobial peptides by model membranes as well as the perturbation that they induce in lipid packing. In particular, we focused on some peptides that have simultaneously acidic and basic residues. The net charges of these peptides are modulated by pH changes and the lipid composition of model membranes. We discuss the experimental approaches used to explore these aspects of lipid membranes using lipid vesicles and lipid monolayer as model membranes.     

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in β cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.     

Particle Transport through Hydrogels Is Charge Asymmetric

Transport processes within biological polymer networks, including mucus and the extracellular matrix, play an important role in the human body, where they serve as a filter for the exchange of molecules and nanoparticles. Such polymer networks are complex and heterogeneous hydrogel environments that regulate diffusive processes through finely tuned particle-network interactions. In this work, we present experimental and theoretical studies to examine the role of electrostatics on the basic mechanisms governing the diffusion of charged probe molecules inside model polymer networks. Translational diffusion coefficients are determined by fluorescence correlation spectroscopy measurements for probe molecules in uncharged as well as cationic and anionic polymer solutions. We show that particle transport in the charged hydrogels is highly asymmetric, with diffusion slowed down much more by electrostatic attraction than by repulsion, and that the filtering capability of the gel is sensitive to the solution ionic strength. Brownian dynamics simulations of a simple model are used to examine key parameters, including interaction strength and interaction range within the model networks. Simulations, which are in quantitative agreement with our experiments, reveal the charge asymmetry to be due to the sticking of particles at the vertices of the oppositely charged polymer networks.     

The antibiotic activity of cationic linear amphipathic peptides: lessons from the action of leucine/lysine copolymers on bacteria of the class Mollicutes.

Peptides composed of leucyl and lysyl residues ('LK peptides') with different compositions and sequences were compared for their antibacterial activities using cell wall-less bacteria of the class Mollicutes (acholeplasmas, mycoplasmas and spiroplasmas) as targets. The antibacterial activity of the amphipathic alpha-helical peptides varied with their size, 15 residues being the optimal length, independent of the membrane hydrophobic core thickness and the amount of cholesterol. The 15-residue ideally amphipathic alpha helix with a +5 positive net charge (KLLKLLLKLLLKLLK) had the strongest antibacterial activity, similar to that of melittin. In contrast, scrambled peptides devoid of amphipathy and the less hydrophobic beta-sheeted peptides [(LK)nK], even those 15-residue long, were far less potent than the helical ones. Furthermore, the growth inhibitory activity of the peptides was correlated with their ability to abolish membrane potential. These data are fully consistent with a predominantly flat orientation of LK peptides at the lipid/water interface and strongly supports that these peptides and probably the linear polycationic amphipathic defence peptides act on bacterial membranes in four main steps according to the 'carpet' model: (a) interfacial partitioning with accumulation of monomers on the target membrane (limiting step); (b) peptide structural changes (conformation, aggregation, and orientation) induced by interactions with the lipid bilayer (as already shown with liposomes and erythrocytes); (c) plasma membrane permeabilization/depolarization via a detergent-like effect; and (d) rapid bacterial cell death if the extent of depolarization is maintained above a critical threshold.     

Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides

Synthetic peptides of the five alternative NH2-terminal sequences of Shaker when applied to the cytoplasmic side of ShB channels that have an NH2-terminal deletion (ShB delta 6-46) block the channel with potencies correlated with the rate of inactivation in the corresponding variant. These peptides share no sequence similarity and yet three out of the five have apparent dissociation constants between 2 and 15 microM, suggesting that the specificity requirements for binding are low. To identify the primary structural determinants required for effective block of ShB delta 6-46, we examined the effects of substitutions made to the 20 residue ShB peptide on association and dissociation rates. Nonpolar residues within the peptide appear to be important in stabilizing the binding through hydrophobic interactions. Substitutions to leucine-7 showed there was a clear correlation between hydrophobicity and the dissociation rate constant (koff) with little effect on the association rate constant (kon). Substituting charged residues for hydrophobic residues within the region 4-8 disrupted binding. Within the COOH-terminal half of the peptide, substitutions that increased the net positive charge increased kon with relatively small changes in koff, suggesting the involvement of long-range electrostatic interactions in increasing the effective concentration of the peptide. Neutralizing charged residues produced small changes in koff. Charges within the region 12-20 act equivalently; alterations which conserved net charge produced little effect on either kon or koff. The results are consistent with this region of the peptide having an extended conformation and suggest that when bound this region makes few contacts with the channel protein and remains relatively unconstrained. Analogous mutations within the NH2-terminal domain of the intact ShB channel produced qualitatively similar effects on blocking and unblocking rates.     

Hydrophilic Linkers and Polar Contacts Affect Aggregation of FG Repeat Peptides

Transport of large proteins into the nucleus involves two events, binding of the cargo protein to a transport receptor in the cytoplasm and passage of the cargo-transporter complex through the selective permeability barrier of the nuclear pore complex. The permeability barrier is formed by largely disordered polypeptides, each containing a number of conserved hydrophobic phenylalanine-glycine (FG) sequence motifs, connected by hydrophilic linkers of varying sequence (FG nups). How the motifs interact to form the permeability barrier, however, is not yet known. We have, therefore, carried out molecular dynamics simulations on various model FG repeat peptides to study the aggregation propensity of FG nups and the specific roles of the hydrophobic FG motifs and the hydrophilic linkers. Our simulations show spontaneous aggregation of the model nups into hydrated aggregates, which exhibit structural features assumed to be part of the permeability barrier. Our simulations suggest that short β-sheets are an important structural feature of the aggregates and that Phe residues are sufficiently exposed to allow rapid binding of transport receptors. A surprisingly large influence of the amino acid composition of the hydrophilic linkers on aggregation is seen, as well as a major contribution of hydrogen-bonding patterns.     

Assessment of RNA carrier function in peptide amphiphiles derived from the HIV fusion peptide

A small library of amphiphilic peptides has been evaluated for duplex RNA carrier function into A549 cells. We studied peptides in which a C-terminal 7-residue cationic domain is attached to a neutral/hydrophobic 23-residue domain that is based on the viral fusion peptide of HIV. We also examined peptides in which the cationic charge was evenly distributed throughout the peptide. Strikingly, subtle sequence variations in the hydrophobic domain that do not alter net hydrophobicity result in wide variation in RNA uptake. Additionally, cyclic cystine variants are much less active as RNA carriers than their open-chain cysteine analogs. With regard to electrostatic effects, we find that lysine is less effective than arginine in facilitating uptake, and that even distribution of cationic residues throughout the peptide sequence results in especially effective RNA carrier function. Overall, minor changes in peptide hydrophobicity, flexibility and charge distribution can significantly alter carrier function. We hypothesize this is due to altered properties of the peptide-RNA assembly rather than peptide secondary structure.     

Transport of polymeric nanoparticle gene carriers in gastric mucus

Nanoparticle transport through mucosal barriers is often restricted owing to mucoadhesion and the highly viscoelastic nature of mucus gels, which may limit efficient drug and gene delivery. We formulated sub-200 nm particulates from poly(d,l-lactic-co-glycolic) acid (PLGA) and the cationic surfactant dimethyldioctadecylammonium bromide (DDAB). Subsequently, anionic DNA was condensed to the surface to obtain gene carriers with transfection rates 50-fold higher than those of naked DNA in vitro. Using the method of multiple particle tracking (MPT), we measured the transport rates of dozens of individual PLGA-DDAB/DNA nanoparticles in real time in reconstituted pig gastric mucus (PGM) that possessed physiologically relevant rheological properties. The average transport rate of PLGA-DDAB/DNA nanoparticles was 10-fold higher than those of similar size polystyrene nanoparticles. Improved transport rates, stability in mucus, and ability to transfect cells make PLGA-DDAB/DNA nanoparticles candidates for mucosal DNA vaccines and gene therapy.     

Membrane association, electrostatic sequestration, and cytotoxicity of Gly-Leu-rich peptide orthologs with differing functions

The skins of closely related frog species produce Gly-Leu-rich peptide orthologs that have very similar sequences, hydrophobicities, and amphipathicities but differ markedly in their net charge and membrane-damaging properties. Cationic Gly-Leu-rich peptides are hemolytic and very potent against microorganisms. Peptides with no net charge have only hemolytic activity. We have used ancestral protein reconstruction and peptide analogue design to examine the roles of electrostatic and hydrophobic interactions in the biological activity and mode of action of functionally divergent Gly-Leu-rich peptides. The structure and interaction of the peptides with anionic and zwitterionic model membranes were investigated by circular dichroism with 2-dimyristoyl-sn-glycero-3-phosphatidylcholine or 1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol vesicles and surface plasmon resonance with immobilized bilayers. The results, combined with antimicrobial assays, the kinetics of bacterial killing, and membrane permeabilization assays, reveal that Gly, Val, Thr, and Ile can all be accommodated in an amphipathic alpha helix when the helix is in a membrane environment. Binding to anionic and zwitterionic membranes fitted to a 2-stage interaction model (adsorption to the membrane followed by membrane insertion). The first step is governed by hydrophobic interactions between the nonpolar surface of the peptide helix and the membranes. The strong binding of Gly-Leu-rich cationic peptides to anionic membranes is due to the second binding step and involves short-range Coulombic interactions that prolong the residence time of the membrane-inserted peptide. The data demonstrate that evolution has positively selected charge-altering nucleotide substitutions to generate an orthologous cationic variant of neutral hemolytic peptides that bind to and permeate bacterial cell membranes.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Interactions of calmodulin with death-associated protein kinase peptides: experimental and modeling studies

We have studied the interactions between calmodulin (CaM) and three target peptides from the death-associated protein kinase (DAPK) protein family using both experimental and modeling methods, aimed at determining the details of the underlying biological regulation mechanisms. Experimentally, calorimetric binding free energies were determined for the complexes of CaM with peptides representing the DAPK2 wild-type and S308D mutant, as well as DAPK1. The observed affinity of CaM was very similar for all three studied peptides. The DAPK2 and DAPK1 peptides differ significantly in sequence and total charge, while the DAPK2 S308D mutant is designed to model the effects of DAPK2 Ser308 phosphorylation. The crystal structure of the CaM-DAPK2 S308D mutant peptide is also reported. The structures of CaM-DAPK peptide complexes present a mode of CaM-kinase interaction, in which bulky hydrophobic residues at positions 10 and 14 are both bound to the same hydrophobic cleft. To explain the microscopic effects underlying these interactions, we performed free energy calculations based on the approximate MM-PBSA approach. For these highly charged systems, standard MM-PBSA calculations did not yield satisfactory results. We proposed a rational modification of the approach which led to reasonable predictions of binding free energies. All three complexes are strongly stabilized by two effects: electrostatic interactions and buried surface area. The strong favorable interactions are to a large part compensated by unfavorable entropic terms, in which vibrational entropy is the largest contributor. The electrostatic component of the binding free energy followed the trend of the overall peptide charge, with strongest interactions for DAPK1 and weakest for the DAPK2 mutant. The electrostatics was dominated by interactions of the positively charged residues of the peptide with the negatively charged residues of CaM. The nonpolar binding free energy was comparable for all three peptides, the largest contribution coming from the Trp305. About two-thirds of the buried surface area corresponds to nonpolar residues, showing that hydrophobic interactions play an important role in these CaM-peptide complexes. The simulation results agree with the experimental data in predicting a small effect of the S308D mutation on CaM interactions with DAPK2, suggesting that this mutation is not a good model for the S308 phosphorylation.