Current Topics: Reviews
Flip-Flop Promotion Mechanisms by Model Transmembrane Peptides
2022 Volume 70 Issue 8 Pages 519-523
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2022 Volume 70 Issue 8 Pages 519-523
Lipid transbilayer movement (flip-flop) is regulated by membrane proteins that are involved in homeostasis and signaling in eukaryotic cells. In the plasma membrane, an asymmetric lipid composition is maintained by energy-dependent unidirectional transport. Energy-independent flip-flop promotion by phospholipid scramblases disrupts the asymmetry in several physiological processes, such as apoptosis and blood coagulation. In the endoplasmic reticulum, rapid flip-flop is essential for bilayer integrity because phospholipids are synthesized only in the cytoplasmic leaflet. Phospholipid scramblases are also involved in lipoprotein biogenesis, autophagosome formation, and viral infection. Although several scramblases have been identified and investigated, the precise flip-flop promotion mechanisms are not fully understood. Model transmembrane peptides are valuable tools for investigating the general effects of lipid–peptide interactions. We focus on the development of model transmembrane peptides with flip-flop promotion abilities and their mechanisms.
Phospholipids are amphiphilic molecules composed of hydrophilic headgroups and hydrophobic acyl chains. Their amphiphilic nature allows them to spontaneously form lipid bilayers that are the fundamental structures of biological membranes. Lipid bilayers in the liquid crystalline phase are dynamic entities.1,2) The time scales of phospholipid movements in the bilayers, such as lateral diffusion and acyl chain motion, range from pico- to nano-seconds,3–9) giving biological membranes a fluid nature. Fluidity allows dynamic interactions between proteins and surrounding molecules in signaling processes and membrane fusion/fission in vesicular transport and proliferation. In contrast, lipid transbilayer movement (flip-flop) is extremely slow (hours to days) in artificial lipid bilayers10–14) owing to the energy barrier for phospholipid headgroups to transverse hydrophobic hydrocarbon regions. Therefore, in biological membranes, phospholipid flip-flop is promoted by proteins in an energy-dependent or -independent manner. Phospholipid scramblases mediate energy-independent flip-flop promotion. Although the three-dimensional (3D) structures of scramblases have already been reported,15–17) flip-flop promotion mechanisms still need to be investigated.18) Model membrane systems enable us to investigate the general effects of proteins on lipid movement. Moreover, the development of functionalized peptides leads to the control of cell functions and treatment of diseases. In this review, we focus on the development of model transmembrane peptides with flip-flop promotion abilities and their mechanisms.
First, we briefly review the biological roles of phospholipid flip-flop before discussing model transmembrane peptides. In the plasma membrane (PM) of eukaryotic cells, phospholipids are asymmetrically distributed with the aid of P4-ATPases and ATP-binding cassette (ABC) transporters.19–21) Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are localized in the cytoplasmic leaflet, whereas phosphatidylcholine (PC) and sphingomyelin (SM) are present in the outer leaflet.22) Several P4-ATPases specifically recognize PS and PE, and transport (flip) them from the exoplasmic leaflet to the cytoplasmic leaflet.19,21) ABC transporters in the PM generally act as outward transporters (floppases) with lower substrate specificity; not only PC and SM but also PS and PE are transported.19–21) The localization of PS in the cytoplasmic leaflet is important for the recognition of the PM by several signaling proteins.23,24)
Phospholipid scramblases are activated in various biological processes, resulting in PS exposure on the cell surface.21,24,25) In apoptotic cells, caspase-mediated cleavage activates the PM scramblase, Xkr8, and inactivates P4-ATPases.26,27) Macrophages recognize PS on the surface of apoptotic cells and engulf them.28) Therefore, the exposed PS is referred to as an eat-me-signal. In the blood coagulation process, another PM scramblase, TMEM16F, is activated by an increase in intracellular Ca2+ concentration.29) The exposed PS functions as a scaffold for blood-clotting factors and promotes their complex formation.25) Additionally, PS exposure has been observed in other biological processes, including bone mineralization, viral infection, myoblast fusion, fertilization, erythropoiesis, erythrocyte clearance, rod cell shedding, and synaptic pruning.25)
Phospholipid scrambling also plays a critical role in intracellular membranes. The endoplasmic reticulum (ER) membrane is a major phospholipid synthesis site in cells.30,31) The rapid flip-flop in the ER membrane32–35) is a prerequisite for the integrity of the bilayer structure because phospholipid synthesis occurs mainly in the cytoplasmic leaflet.30,31,36,37) Although ER scramblases have long been enigmatic, TMEM41B and VMP1 have recently been identified as ER-resident scramblases, and their deficiency changes the ER structure and impairs lipoprotein production in liver cells.38–41) Lipoproteins are composed of a triglyceride and cholesterol core, covered by phospholipids and apolipoproteins. Phospholipid scrambling is necessary for access to bulk phospholipids in the ER during lipoprotein growth because lipoproteins are produced in the lumen of the ER.39) TMEM41B and VMP1 also play critical roles in the infection of flaviviruses and coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).42) Although TMEM16K has also been proposed as a lipid scramblase in the ER, further research regarding its biological roles is required.43) ATG9A functions as a phospholipid scramblase in autophagosomes.17) During phagophore expansion, phospholipids are transported from the ER to the outer leaflet of phagophores. Rapid phospholipid scrambling is, therefore, critical for the membrane integrity of phagophores. Indeed, ATG9A-knockout inhibits large autophagosome formation.17,44) Additionally, TMEM41B and VMP1 have also been suggested to regulate autophagosome formation.41,45,46) Constitutive lipid scrambling has also been observed in the disc membranes of retinal rod photoreceptor cells.47–49) One of the proteins responsible for lipid scrambling is the light-sensing protein, rhodopsin.50–53) Although the roles of rhodopsin-mediated lipid scrambling need to be elucidated, rhodopsin may support the function of ABCA4, which is the flippase for PE and N-retinylidene PE.21,54) ABCA4 facilitates removal of the latter lipid that is potentially toxic. However, unidirectional transport results in the accumulation of lipids in the cytoplasmic leaflet and compromises the integrity of the disc membranes. Therefore, rhodopsin may help ABCA4 to function properly by relieving the transbilayer lipid imbalance.
Kol et al. first evaluated the flop of fluorescent lipids using transmembrane peptides with a Leu-Ala repeat sequence and terminal X residues (X = Lys, His, or Trp) known as XALP55) (Table 1). XALP peptides can be incorporated into PC membranes to form transmembrane α-helices.56) All XALP peptides increase the flop rate of phosphatidylglycerol (PG), and KALP and WALP peptides also promote PE flop.55,57) The similar flop-promoting abilities of XALP peptides with varying flanking residues suggest that the mere presence of transmembrane helices is important for phospholipid flip-flop promotion. Local perturbations in the vicinity of the helix may facilitate flip-flop (Fig. 1A, left). Langer et al. demonstrated that distinct α-helical peptides with membrane-spanning sequences composed of Leu, Leu-Val repeats, or Leu-Leu-Val repeats facilitate the flip of PC, PE, and PS58) (Table 1). The highly dynamic helical backbone may disrupt lipid acyl chains and promote flip-flop (Fig. 1A, right). However, the activities of these peptides are significantly lower than those of lipid scramblases, and time-resolved small-angle neutron scattering (TR-SANS) experiments revealed that the KALP23 peptide does not have any effects on 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) flip-flop,13) which is one of the most abundant lipid species in eukaryotic cells. These results motivated us to identify additional factors to enhance phospholipid flip-flop.
| Property | Name | Sequence |
|---|---|---|
| Hydrophobic | KALP23a) | GKKLALALALALALALALALKKA |
| WALP23a) | GWWLALALALALALALALALWWA | |
| HALP23a) | GHHLALALALALALALALALHHA | |
| L16b) | KKKWLLLLLLLLLLLLLLLLKKK | |
| LV16b) | KKKWLVLVLVLVLVLVLVLVKKK | |
| LLV16b) | KKKWLLVLLVLLVLLVLLVLKKK | |
| Hydrophilic residue | TMP-Ec) | GKKLALALALAEWLALALALKKA |
| TMP-Kc) | GKKLALALALAKWLALALALKKA | |
| pL15(D10)d) | KKLLLLLLLDWLLLLLLLLKK | |
| TMP23Qe) | GKKLALALALAQWLALALALKKA | |
| TMP23Ne) | GKKLALALALANWLALALALKKA | |
| TMP23He) | GKKLALALALAHWLALALALKKA | |
| TMP23Pe) | GKKLALALALAPWLALALALKKA | |
| TMP23Ye) | GKKLALALALAYWLALALALKKA | |
| TMP23Se) | GKKLALALALASWLALALALKKA | |
| TMP23Te) | GKKLALALALATWLALALALKKA | |
| 9R1Hf) | WKKLALALRHALALALALALALKK | |
| 9R2Hf) | WKKLALALRLHLALALALALALKK | |
| 9R3Hf) | WKKLALALRLAHALALALALALKK | |
| 9R4Hf) | WKKLALALRLALHLALALALALKK | |
| 9R5Hf) | WKKLALALRLALAHALALALALKK | |
| 9R6Hf) | WKKLALALRLALALHLALALALKK | |
| 9R7Hf) | WKKLALALRLALALAHALALALKK | |
| 9R8Hf) | WKKLALALRLALALALHLALALKK | |
| 9R9Hf) | WKKLALALRLALALALAHALALKK | |
| 10R3Hf) | WKKLALALARALHLALALALALKK | |
| 7R3Hf) | WKKLALRLAHALALALALALALKK | |
| 5R3Hf) | WKKLRLAHALALALALALALALKK | |
| EDEM1g) | KKVLGLVLLRLGLHGVLWLVFGLGPKK | |
| Mismatch | TMP25Qe) | GKKALALALALAQWLALALALAKKA |
| TMP27Qe) | GKKLALALALALAQWLALALALALKKA | |
| TMP31Qe) | GKKLALALALALALAQWLALALALALALKKA |
(A) Hydrophobic transmembrane peptides. (B) Transmembrane peptides with hydrophilic residues. Red regions indicate hydrophilic residues. (C) Transmembrane peptide in negative mismatch.
Charged residues (Lys or Glu) were introduced to the center of the membrane-spanning sequences of the KALP23 derivative peptides59) (TMP-K or -E, Table 1). The conformation of the peptides in membranes were evaluated using fluorescent fatty acids, n-ASs (n = 2, 6, and 12), in which fluorophores locate at varying depths in membranes. The efficiency of fluorescent resonance energy transfer between n-ASs and the Trp residue of the peptides depends on the conformation because the Trp residue lies near the center of the sequences. Despite the presence of charged residues in the transmembrane regions, these peptides have a transmembrane conformation in the PC membrane. TR-SANS experiments demonstrated that both peptides promoted POPC flip-flop. Fluorescence experiments revealed that TMP-E and TMP-K exhibit a high flip-promoting ability for PC and PG, respectively, suggesting that electrostatic interactions between polar lipid headgroups and charged residues in the hydrocarbon region are important for flip-flop promotion. Consistently, LeBarron and London demonstrated the flip-promotion ability for PC of a transmembrane peptide with an Asp residue at the center60) (Table 1).
A more detailed characterization of the effects of hydrophilic residues on flip-flop promotion was performed using a series of transmembrane peptides with varying hydrophilic residues at the center61) (Table 1). The flip promotion abilities of the peptides increase with increasing hydrophilicity of the central residues, and the highest activity is observed for the Gln-containing peptide, TMP23Q. Fluorescence spectra of Trp residues located near the center of the sequences indicate that the polarity in the vicinity of Trp residues increases significantly for the peptides containing a higher hydrophilic residue. This is presumably due to the partitioning of water molecules into the hydrocarbon region, which are known as membrane (water) defects. A positive correlation was observed between the flip-promotion ability of the peptides and the degree of red shift in the Trp fluorescence spectra, suggesting the attenuation of the flip-flop energy barrier by membrane defects. Gurtovenko and Vattulainen used all-atom molecular dynamics (MD) simulations to show that a transient membrane defect induces phospholipid flip-flop.62) The promotion of flip-flop by membrane defects has also been suggested in experimental research.63) Hydrophilic amino acids placed in the hydrocarbon region mediate membrane defects in the vicinity.64,65) Water molecules partition into the hydrocarbon region to stabilize the hydrophilic moieties by hydration. Thus, transmembrane peptides with hydrophilic residues might promote phospholipid flip-flop via membrane defects that can reduce the energy barrier to hydrate lipid headgroups in the hydrocarbon region (Fig. 1B, left).
The relative position and depth of hydrophilic residues have a considerable impact on flip-flop promotion66) (Table 1). The 9RnH peptides have an Arg residue at the 9th position from the N-terminus and a His residue at the nth position from the Arg residue. The flip-promotion ability of the 9RnH peptides changes periodically depending on the turn angle between Arg and His residues, and peptides with Arg and His residues at the approximately same side in the helices (9R3H, 9R4H, and 9R7H) exhibit higher activities. These results indicate that hydrophilic residues on the same side of the helix synergistically enhance phospholipid flip-flop. mR3H peptides were synthesized to evaluate the effect of the depth of hydrophilic residues in the membrane. The 10R3H peptide, which has a His residue at the center of the sequence, retains flip-promotion ability, whereas 5R3H and 7R3H, with hydrophilic residues in a shallower region, have almost no effect on flip-flop. All-atom MD simulations allowed us to obtain a molecular insight into flip-flop promotion by the peptides. The depths of the side chain moieties in hydrophilic residues are clearly different between the flip-promoting (9R3H and 10R3H) and non-promoting (5R3H and 7R3H) peptides. Both hydrophilic residues are located in the hydrocarbon region in the cases of 9R3H and 10R3H, whereas Arg residues of 5R3H and 7R3H have approximately the same distribution as that of carbonyl carbon atoms of phospholipids. This result indicates that hydrophilic residue partitioning in the hydrocarbon region participates in flip-flop promotion by peptides. Interestingly, membrane defects were observed near the flip-promoting peptides in the simulation. The probabilities of lipid headgroups and water molecules closer to the bilayer midplane are increased in the vicinity of the flip-promoting peptides, and are positively correlated with the flip-promoting abilities of the peptides in the experiments. These results also strongly suggest that membrane defects play a critical role in flip-flop promotion by transmembrane peptides with hydrophilic residues (Fig. 1B, right panel). Moreover, a direct interaction between hydrophilic residues and lipids was observed. Hydrogen bonds are formed between the lipid phosphate or carbonyl groups and His or Arg residues in 9R3H. This is consistent with the experimental results for the EDEM1 peptide that has Arg and His residues on the same side of the helix (Table 1), where the decrease in activation free energy for the EDEM1-mediated flip-flop is due to the reduction in activation enthalpy.67) Thus, flip-flop promotion by transmembrane peptides with hydrophilic residues is mediated by direct and indirect (membrane defects) interactions between peptides and lipids.
To date, 3D structures have been resolved for three types of phospholipid scramblases: TMEM16 family, Xkr8, and ATG9. Brunner et al. first presented the 3D structure of a phospholipid scramblase, nhTMEM16.15) They hypothesized that a hydrophilic groove in the transmembrane region is the catalytic site for lipid scrambling. Several experimental studies support this hypothesis68–71) and the translocation of phospholipids passing through the groove has been observed in MD simulations.43,72–74) Xkr8 also has a charged residue cluster aligned parallel to the membrane normal in the transmembrane region.16) Although hydrophilic residues are located in the transmembrane region of ATG9A, they form a water-filled pore in the trimer conformation.17) Thus, hydrophilic residues in transmembrane regions also have critical functions in lipid scrambling by all three types of phospholipid scramblases, albeit with different structures.
3.3. Hydrophobic MismatchHydrophobic mismatch is defined as the difference between the thickness of lipid bilayers and the length of hydrophobic domains of proteins.75) Positive mismatch, which is defined as the length of hydrophobic domains of the peptides being longer than the bilayer thickness, causes membrane thickening and/or tilting of the peptides against the membrane normal to prevent the exposure of the hydrophobic domains to water. On the other hand, membrane thinning and/or peptide clustering occurs in a negative mismatch. TMP27Q and TMP31Q (Table 1) have almost no effect on flip-flop compared with shorter TMP23Q and TMP25Q (Table 1) in the POPC membrane.61) Assuming the length of the α-helix structure per residue is 1.5 Å, the former (TMP27Q and TMP31Q) and latter peptides (TMP23Q and TMP25Q) are in positive and negative mismatches, respectively, suggesting that local membrane thinning in the vicinity of the negative mismatch peptides decreases the energy barrier for flip-flop (Fig. 1C). Therefore, at least for TMPnQ, negative mismatch enhances flip-flop promoting ability of the peptides possibly due to local membrane thinning, whereas positive mismatch has almost no effect on peptide-mediated flip-flop. Membrane thinning was observed near the hydrophilic groove of nhTMEM16, which is one of the possible mechanisms of lipid scrambling.71)
In this review, we introduced flip-flop promotion using the synthesized transmembrane peptides. The presence of hydrophilic residues in the transmembrane region is of critical importance for the flip-flop promotion. Although the lipid scrambling activity of scramblases is significantly higher than that of peptides, artificial lipid scrambling can be utilized to control live cell functions; lipid scrambling in the PM is involved in various physiological processes. Thus, the development of lipid scrambling peptides contributes not only to the understanding of lipid scrambling mechanisms but also to the treatment of diseases by regulating lipid dynamics.
This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers JP16J00095, JP17H02941, JP17H06704, JP19K16086, and JP26287098, the Kao Foundation for Arts and Culture, and the Tokyo Biochemical Research Foundation.
The authors declare no conflict of interest.
