Peptides and anti-Cancer activity
Larry H. Bernstein, MD, FCAP, Curator
LPBI
PE and PS Lipids Synergistically Enhance Membrane Poration by a Peptide with Anticancer Properties
http://www.cell.com/cms/attachment/2035808117/2051293431/gr1.jpgFigure 1
Schematic representation of membrane disruption by peptides and the experimental system. The helical peptide Polybia-MP1 is shown according to the helical wheel projections. Amino acids: (blue) polar with positive net charge; (purple) polar with negative net charge; (red) polar noncharged; and (green) nonpolar. Confocal microscopy was performed to investigate the influx of three dyes with distinct sizes in GUVs in the presence and absence of PE lipids: 0.37 kDa CF (green), 3k-CB (blue), 10k-AF647 (magenta), and the scale bars correspond to 10 μm. Lipid membranes are labeled with Rh-DOPE (red). The peptide interacts with the GUVs, disturbs their structure, and then enables the passage of fluorescent dyes by formation of pore-like structures. To see this figure in color, go online.
http://www.cell.com/cms/attachment/2035808117/2051293432/gr2.jpg
Figure 2
Binding isoterms show that MP1 has a higher affinity for PS-containing membranes. The binding isoterms and the partition coefficients (Kp) obtained using CD by lipid titration at 10 μM MP1 solution. LUVs are composed of (a) PC, (b) PC/PE, (c) PC/PS, and (d) PC/PE/PS.
http://www.cell.com/cms/attachment/2035808117/2051293428/gr4.jpg
Figure 4
Fluorescence requenching assays for MP1 reveal all-or-none leakage in the four lipid compositions studied. Qin is constant as a function of fout for MP1, which is in agreement with the all-or-none mechanism of dye release.) (Lines) Theoretical curves for ideal graded and all-or-none dye release (18). To see this figure in color, go online.
Fig. 4 shows that the values of Qin remain constant with the increase of fout and the consequent increase of peptide/lipid molar ratios. This clearly shows that MP1 exhibits the all-or-none leakage mechanism for all lipid compositions studied, which is in contrast to what has been observed for antimicrobial peptides mastoparan X and mastoparan MP (19). We propose that this all-or-none leakage is related to peptide-induced pore formation (20, 21, 22, 23), where the vesicles are able to release all their internal contents through pore-like structures that are sufficiently long lived (23, 24, 25, 26, 27, 28). We do not solely attribute the all-or-none leakage to lysis of the vesicles because nonlysed, leaky vesicles are observed in our GUV experiments (Figs. 3 and S1). However, we do not discount the possibility that lysis might play a role in the LUV leakage at the highest peptide concentrations used in this assay. Furthermore, pore-like activity of MP1 has previously been identified from electrophysiology measurements in planar lipid bilayers composed of phytanoyl-PC and phytanoyl-PC/PS (70:30) (4).
20% (unleaked) or 
80% (fully leaked) leakage (Fig. S2).Introduction
Materials and Methods
Materials
Peptide synthesis and purification
Mass spectrometry analysis
GUV formation
LUV preparation
CD spectroscopy for binding isotherms
Confocal microscopy
Analysis of confocal images and movies
ANTS/DPX requenching measurements
AFM
Phase contrast microscopy
Results
PS lipids significantly enhance peptide binding to the membrane
MP1 dose-response studies reveal that PE and PS lipids enhance membrane permeability at lower peptide concentrations
Confirmation of the pore-formation hypothesis in lipid vesicles
Synergistic enhancement of GUV leakage kinetics by PE and PS lipids
PE lipids significantly enhance pore size and membrane permeability
Direct imaging of peptide-induced pores by AFM
Discussion
Biophysical implications for MP1-lipid membrane interactions
Implications for the chemotherapeutic potential of MP1 peptides
Author Contributions
Supporting Material
References
Synergistic enhancement of GUV leakage kinetics by PE and PS lipids
Analysis of the time delay from the start of our GUV experiments (addition of the peptide) to observations of the onset of GUV leakage reveals a synergistic reduction in this lag time for PC/PE/PS membranes (Table 1 and Fig. 5). In these GUV experiments, we add 4.0 μM MP1 to our samples and monitor the time taken for initial leakage events of GUVs to the 0.37 kDa CF probe to occur (t0 − tCF). This MP1 concentration is chosen as it causes significant leakage of GUVs within 30 min of peptide addition across all four lipid compositions of interest. The onset of leakage occurs approximately twice as quickly for PC/PE/PS GUVs than for other membrane compositions, with only a very slight reduction of the lag time for PC/PS membranes compared with PC/PE and PC GUVs. Therefore, this is not a purely electrostatic effect from the increased rate and extent of peptide binding to anionic PS-containing membranes; it also requires the presence of PE to significantly increase the susceptibility of the membrane to permeabilization.
| Time Delays (s) | PC/PE | PC/PE/PS | PC/PS | PC |
|---|---|---|---|---|
| t0 − tCF | 1600 ± 110 | 760 ± 120 | 1400 ± 60 | 1600 |
| tCF – t3k-CB | 1.8 ± 0.6 | 1.5 ± 0.3 | 41 ± 5a | 160 ± 110 |
| tCF – t10k-AF647 | 4.2 ± 1.5 | 2.0 ± 0.4 | 52 ± 6 | 220 ± 66 |
| t3k-CB – t10k-AF647 | 2.7 ± 0.9 | 1.4 ± 0.4 | 9.6 ± 1.2 | 60 ± 42 |
The errors represent the standard deviation of the observed GUV data set.
http://www.cell.com/cms/attachment/2035808117/2051293430/gr6.jpg
Figure 6
PE lipids facilitate much greater membrane permeability in GUV membranes. (a) Typical log-linear plot of time-dependent dye influx: −R/3ln(1−c) (10−5) versus time, for the three dyes in a single GUV of PC/PS. (b) Distributions of the obtained permeabilities in single GUVs composed of PC/PS. (c) Typical log-linear plot of time-dependent dye influx: −R/3ln(1−c) (10−5) versus time, for the three dyes in a single GUV of PC/PE/PS. (d) Distributions of the obtained permeabilities in single GUVs composed of PC/PE/PS. The permeabilities are obtained from the slopes of the log-linear plots of the time-dependent influx of dyes into single GUVs. To see this figure in color, go online.
Our leakage kinetic profiles were used to calculate the membrane permeability to the different-sized probes using a diffusional model for membrane translocation (29); the membrane permeability is the gradient of the log-linear plot as seen in the example data in Fig. 6, a and c. Average permeability values for each membrane composition to each probe size during the initial leakage events are shown in Table 2. It can be seen that, for all membrane compositions tested, average permeability decreases with increasing probe size. However, the most significant finding from this data is the large, one-order-of-magnitude increase, in membrane permeability for membrane compositions containing 10% PE. This can be observed for all three leakage markers studied. It can also be seen that the presence of PS in the membrane imparts a modest, but significant, increase in permeability on the membranes upon perturbation by MP1. This effect can be seen further in Fig. 6, b and d, which show the distributions of permeability measurements for PC/PS and PC/PE/PS GUVs to the CF and 10k-AF647 leakage markers, respectively. For both probe sizes, the majority of permeability measurements for PC/PS membranes were in the 0–25 nm/s range, whereas when PE was included in the membrane formulations, a large proportion of permeability measurements were >500 nm/s.
| 〈Pm〉CF(nm/s) | 〈Ap〉/〈Av〉CF(10−6) | 〈Pm〉3k-CB(nm/s) | 〈Ap〉/〈Av〉3k-CB(10−6) | 〈Pm〉10k-AF647(nm/s) | 〈Ap〉/〈Av〉10k-AF647 (10−6) | |
|---|---|---|---|---|---|---|
| PC | 46 ± 14 | 0.45 ± 0.14 | 17 ± 6 | 0.50 ± 0.16 | 8 ± 2 | 0.44 ± 0.08 |
| PC/PS | 59 ± 12 | 0.58 ± 0.12 | 29 ± 6 | 0.90 ± 0.20 | 23 ± 4 | 1.30 ± 0.20 |
| PC/PE | 466 ± 143 | 4.60 ± 1.40 | 207 ± 102 | 6.50 ± 3.40 | 158 ± 53 | 8.80 ± 2.90 |
| PC/PE/PS | 589 ± 142 | 5.80 ± 1.40 | 333 ± 73 | 10.40 ± 2.80 | 169 ± 52 | 9.40 ± 2.90 |
The errors represent the standard deviation of the observed GUV data set.
It should be noted that the observed permeability distributions (Fig. 6, b and d) are broad due to the fact that peptide-induced pores do not have well-defined structures, pore formation events are stochastic, and the membrane interfaces are fluid, giving rise to this wide distribution of individual permeability events when measured at the single vesicle level. Indeed, it has previously been reported that the initial pores that form during peptide-induced pore formation might be far from equilibrium and can, for example, relax to a smaller size over longer timescales as has been observed for the peptides Bax-α5 (23) and magainin 2 (30).
The permeability data was used to calculate the effective fractional permeable area of the membrane for each probe size using the expression (29)
where Ap is the permeable area of membrane on a GUV, Av is the total area of the vesicle, Pm is the permeability, and δ is the thickness of the membrane. The Stokes-Einstein diffusion constant of the leakage marker is D0 = kT/6πηR0, where kT is the thermal energy, η is the solvent viscosity, and R0 is the hydrodynamic radius of the fluorescent dye that was estimated with the relationR0 = 0.0332(Mw)0.463 in nanometers (31); Mw is the molecular weight of the dye. A brief derivation of this equation is presented in the Supporting Material. It should be noted that this equation is most accurate for the formation of large membrane pores as it assumes that the diffusion constant of the dye within the pore is the same as its diffusion constant in bulk solution. However, we believe this to be a reasonable assumption because these passive leakage markers will have a very short residence time within the pore itself due to the bilayer only being ∼5-nm thick; these solutes are not expected to interact strongly with the membrane itself.
Values of the fractional permeable areas are shown in Table 2. The fractional permeable areas were also found to be an order-of-magnitude greater for membrane compositions containing PE than for those that did not. Note that slightly larger permeable areas were measured for the larger leakage markers; these represent a later time point in the membrane disruption of GUVs by MP1 as the smaller leakage markers translocate the membrane at earlier times (Table 1). This extended delay time therefore allows for a greater area of membrane disruption to occur before the initiation of leakage to the larger Mw dyes.
Besides the order-of-magnitude increase in membrane permeabilization in the presence of PE lipids, we found an interesting correlation between PE content and membrane morphological response to MP1. Without PE, PC and PC/PS GUVs exhibited bright spots of fluorescent lipids at specific locations on the membrane surface in the presence of 4.0 μM MP1 (Fig. 7). We attribute these observations to local aggregation of peptides and lipids at the GUV surface. These peptide-induced lipid aggregates may be in competition with the pore-/defect-forming activity of the peptides. Such dense lipid structures were not seen on GUVs containing PE (PC/PE and PC/PE/PS) upon introduction of the peptide. Therefore, we speculate that the PE suppresses the intramembrane lipid aggregation by more easily facilitating the poration of the membrane.
Discussion
Introduction
Materials and Methods
Materials
Peptide synthesis and purification
Mass spectrometry analysis
GUV formation
LUV preparation
CD spectroscopy for binding isotherms
Confocal microscopy
Analysis of confocal images and movies
ANTS/DPX requenching measurements
AFM
Phase contrast microscopy
Results
PS lipids significantly enhance peptide binding to the membrane
MP1 dose-response studies reveal that PE and PS lipids enhance membrane permeability at lower peptide concentrations
Confirmation of the pore-formation hypothesis in lipid vesicles
Synergistic enhancement of GUV leakage kinetics by PE and PS lipids
PE lipids significantly enhance pore size and membrane permeability
Direct imaging of peptide-induced pores by AFM
Discussion
Biophysical implications for MP1-lipid membrane interactions
Implications for the chemotherapeutic potential of MP1 peptides
Author Contributions
Supporting Material
References
Biophysical implications for MP1-lipid membrane interactions
We have shown a synergistic enhancement of the rate and extent of membrane permeabilization by MP1 peptides when PE and PS lipids are present in the lipid membrane. This picture is confirmed and corroborated by complementary experiments using three different model membrane systems: LUVs, GUVs, and planar-supported bilayers. We consider the perturbation of the membrane by MP1 peptides in two steps: 1) binding of the peptides to the membrane, and 2) perturbation of the bilayer structure by bound peptides to induce leakage.
Binding isotherms (Fig. 2) reveal that PS lipids cause a 7–8-fold increase in peptide bound to the membrane. This strongly outweighs the small ∼10% reduction in bound peptide concentration caused by the PE lipids. Therefore, we find that the dominant role of PS lipids’ contribution to the membrane disruption by MP1 is a large increase in peptide binding to the membrane.
The role of PE lipids in MP1-induced membrane disruption is twofold: 1) PE increases the susceptibility of the membrane to permeabilization by bound peptides, and 2) PE facilitates the formation of larger transmembrane pores. First, when the extent of GUV leakage is normalized to bound peptide concentration in the dose-response curves in Fig. 3, c and d, it can be seen that 4–5 times lower bound peptide concentration is required to induce a similar leakage response compared to comparable GUVs without PE lipids. Second, GUV and AFM experiments corroborate the effect of larger pores forming in the presence of PE. Quantitative analysis of GUV leakage profiles in Fig. 6 andTable 2 reveal that the presence of PE increases the permeability of membranes by an order of magnitude compared to membranes lacking in PE. Furthermore, once pores formed in GUVs, they quickly (within seconds) grew large enough in size to allow larger macromolecules (3 and 10 kDa) to permeate the membrane (Table 1); this compared to several tens of seconds for larger pores to form in GUVs lacking PE. Crucially, the formation of larger pores for PE-containing membranes is directly visualized by AFM (Fig. 8), where the observed pore diameters are ∼5 times larger in the presence of PE (and hence ∼20–30 times larger in average pore area, consistent with the order-of-magnitude increase in permeability reported for the GUVs).
The formation of transmembrane pores was confirmed by complementary experimental systems and techniques. Rapid translocation of membrane-impermeable leakage markers across GUV membranes, an all-or-none LUV fluorescence leakage assay, and direct visualization of transmembrane defects by AFM imaging of planar bilayers, all confirm this to be true. While these pores are fairly long lived, the membranes were sometimes observed to temporarily reseal, regaining their barrier properties. This can clearly be seen in the leakage profiles of individual GUVs in Figs. 5a, S4, and S5. GUV and planar bilayer imaging experiments also strongly suggest differences in the mechanism of pore formation depending on whether PE lipids are present. Images of GUVs that did not contain PE lipids often exhibited bright spots of increased local lipid concentrations on the membrane, which we interpret to be local aggregation of peptides and lipid (Fig. 7). Similarly, AFM images showed locally raised regions of lipid scattered across the membrane for these lipid compositions (Fig. S9) before the formation of pores (Fig. S10). This contrasted to the pore-formation mechanism observed in the presence of PE, where local aggregates were not directly observed on the GUV surface and time-resolved AFM imaging showed pore growth to occur by the stepwise micellization and loss of lipid from the edge of the pores (Fig. S8).
Besides the increased binding due to PS and the increased membrane susceptibility and pore size due to PE, the synergistic enhancement of membrane disruption facilitated by these lipids can be observed in the kinetics of initial permeabilization events. GUV experiments showed that PC/PE/PS GUVs leaked a factor-of-two quicker than other membrane compositions (Table 1). This is again corroborated by the AFM studies where defects were observed in PC/PE/PS membranes almost immediately after peptide addition, whereas perturbations of other membrane compositions took a few 10 s of minutes to evolve. The complementary pore-promoting effects of PS on bound peptide concentrations and PE on membrane susceptibility far outweigh their slight inhibitory effects on each other’s roles (PE causes a slight reduction in binding affinity (Fig. 2) and PS causes a decrease in the membrane susceptibility to bound peptide (Fig. 3, c and d)). This is apparent from the effects of MP1 on GUVs, where PC/PE/PS membranes experience the greatest membrane perturbation for any given total peptide concentration (Fig. 3, a and b) and the larger number of pores observed on the membrane surface by AFM (Fig. 8). Therefore, our combined results provide a detailed mechanistic picture of the importance of increased PS and PE lipid concentrations in synergistically enhancing the membrane’s propensity for significant disruption of its barrier properties by MP1 peptides.
Variations in lipid composition are responsible for differences in membrane properties such as charge, fluidity, lateral pressure profiles, and mechanical moduli. Changes in these fundamental membrane properties directly affect their interactions with extraneous compounds, such as antimicrobial peptides. The cationic nature of the MP1 peptide is likely an important component in the initial step of peptide action, in which the peptide recognizes the target membrane due to electrostatic interactions and binds to it in a structured form, most of the time as a helix. Therefore, the inclusion of anionic PS lipids in these membranes increases these electrostatic interactions with the MP1 peptide (net charge = +2e). However, MP1-membrane interactions cannot be solely driven by electrostatics as these peptides were also found to disrupt zwitterionic PC and PC/PE membranes, likely through secondary hydrophobic initial binding interactions that lead to a significantly lower bound concentration of peptide compared to the anionic membranes.
Next, insertion of the peptide into the bilayer likely takes place due to the hydrophobic effect, where nonpolar MP1 residues insert into the bilayer core, and defects may then be opened within the membrane structure, leading to its disruption. Furthermore, taking account of the fact that MP1 is a short peptide (14 amino acids) and hence not long enough to form a bilayer-spanning barrel stave pore (9, 32), we anticipate that these pores will be disorganized toroidal structures formed by lipids and peptides, as described by many molecular-dynamics studies (33, 34). PE is known to significantly modulate the lateral pressure profile through membranes and thereby induce negative curvature stress in the bilayer. Negative curvature stress has been shown to enhance the formation of toroidal lipid pores within a membrane by stabilizing the curvature of these structures (35). Therefore, PE would be expected to favor the formation of pore-like defects in the membrane, consistent with the increase susceptibility of these membranes to MP1-induced poration and the order-of-magnitude increase in membrane permeability that we find for PE-containing membranes upon interaction with MP1 peptides.
Introduction
Materials and Methods
Materials
Peptide synthesis and purification
Mass spectrometry analysis
GUV formation
LUV preparation
CD spectroscopy for binding isotherms
Confocal microscopy
Analysis of confocal images and movies
ANTS/DPX requenching measurements
AFM
Phase contrast microscopy
Results
PS lipids significantly enhance peptide binding to the membrane
MP1 dose-response studies reveal that PE and PS lipids enhance membrane permeability at lower peptide concentrations
Confirmation of the pore-formation hypothesis in lipid vesicles
Synergistic enhancement of GUV leakage kinetics by PE and PS lipids
PE lipids significantly enhance pore size and membrane permeability
Direct imaging of peptide-induced pores by AFM
Discussion
Biophysical implications for MP1-lipid membrane interactions
Implications for the chemotherapeutic potential of MP1 peptides
Author Contributions
Supporting Material
References
Implications for the chemotherapeutic potential of MP1 peptides
The MP1 peptide has been shown to have selective inhibition against numerous cancer lines compared to healthy cells (2, 3). Such malignant cells are also known to have increased expression of PS and PE lipids on their outer plasma membrane (5, 6, 7). This study strongly correlates the enhanced tumor inhibitory effects of these peptides with this pathological change in plasma membrane lipid composition, where the upregulation of PS and PE lipids can synergistically enhance the membrane-permeabilizing activity of MP1. This membrane permeabilization is likely to be the primary mechanism of cancer cell death induced by these peptides.
This suggests that MP1 might be a candidate therapeutic for development of novel cancer therapies, or at least guide the development of novel lead compounds for treatment of these diseases. One challenge for the application of antimicrobial peptides in medicine is that they often do not show high enough selectivity to their target cells to result in a favorable therapeutic index for these compounds (36). However, MP1 does not exhibit hemolytic activity to rat erythrocytes but presents chemotaxis for polymorphonucleated leukocytes and potent antimicrobial action against Gram-positive and Gram-negative bacteria (12), suggesting it could have favorable selectivity. It may also be of interest to test MP1 in a combination therapy with other chemotherapeutics that have intracellular targets. The selectivity of the MP1 peptide to disrupt the membranes of cancer cells may act synergistically with these other drugs to significantly enhance the therapeutic potency. Therefore, the therapeutic potential of this and other membrane-active peptides within the field of oncology is worthy of further investigation.
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