4 SCRAMBLASES AND LIPID FLIP–FLOP
The second part of this Thesis focuses on another type of signaling where dysfunc- tioning proteins are also at the root of several serious diseases. Cellular signaling is often dependent on a certain lipid composition on the outer side of a cellular bi- layer. This lipid composition is regulated by specific types of proteins known as flippases and scramblases. Their function is to facilitate lipid translocation across the membrane interior, which is intrinsically a rare event. In addition to cellular signaling, rapid lipid translocation is necessary for other cellular activities as well.
Failure to transport lipids from one side of the bilayer to the other, or transport of the wrong types of lipids, usually has severe consequences for the cell. In Paper IV of this Thesis we present extensive discussion on the roles lipids have in cellular sig- naling. The author contributed to the publication by studying the scramblase prop- erties of rhodopsin and the mechanisms with which it transports lipids across the bilayer. In this chapter, we discuss transbilayer lipid motion, the roles of scramblase proteins and the potential diseases that occur if this process is somehow hindered.
Lipid transfer between cellular components (from one bilayer to the other) is gen- erally achieved with the help of transporter proteins or by exocytosis and endocyto- sis – mechanisms by which lipids can exit and enter a bilayer surface. One example emphasizing the importance of lipid transfer is the sustainability of the plasma mem- brane surrounding the cell. The majority of all cellular lipids are synthesized in two sites within a cell: the endoplasmic reticulum and the Golgi apparatus[97]. In order to preserve cellular integrity, lipids synthesized in these two locations must be trans- ferred to the plasma membrane, which is unable to create new lipids by itself. This is achieved by transporting lipids across the cytoplasm of the cell to the cytosolic side of the plasma membrane. From there, half of the transported lipids must transfer across the bilayer onto the cell surface in order to balance the number of lipids on both leaflets. This transbilayer lipid motion is often referred to as lipid flip–flop.
Lipid flip–flop has major importance in cellular physiology. It is vital for sustain- ing the asymmetric nature of many biological membranes. As mentioned before, phospholipid synthesis occurs only on the cytoplasmic side of the endoplastic retic- ulum. For the ER to grow in a proper manner, half of the synthesized lipids must flip–flop across the ER bilayer to the lumenal side[98]. Flip–flops are also neces- sary to sustain the asymmetrical lipid compositions of various cellular organelles [99, 100]. For instance, the curvature of a membrane increases when lipids with smaller headgroups on the inner leaflet oppose lipids with larger headgroups on the outer leaflet, signaling a target for scaffolding proteins to attach to, resulting in ex- ocytosis[101]. Programmed cell death, or apoptosis, also occurs due to lipid asym- metry. When a high concentration of phosphatidylserine appears at the surface of a cell, it serves as a signal for phagocytes to destroy the sick cell in a controlled fashion [3, 102].
The mechanisms behind lipid flip–flop are very different from lateral lipid mo- tion occurring in a bilayer due to the amphipathic nature of lipids. The amphi- pathic nature is also the reason why cellular membranes take their form with the hydrophilic headgroups of the lipids forming bonds with water molecules on the outside of a lipid bilayer and the hydrophobic tails facing the inside of the bilayer where they are not in contact with water[103]. In physiological temperatures, the structure of a lipid bilayer can be considered a fluid where lipids and embedded pro- teins are able to traverse laterally with relative ease. Lateral diffusion is energetically very easy as the polar headgroups of the lipids are able to remain in constant contact
with water and there are no clear energetic barriers blocking the motion.
Transverse diffusion, or lipid flip–flop, is another matter entirely. For a lipid to cross the membrane interior, its polar headgroup must first deattach itself from the surrounding water molecules and other lipid headgroups, and enter the hydrophobic core of the membrane, from where it will eventually reach the water located on the other side of the membrane. The lipid tails must also turn around in the process, moving against the lateral flow of the structure. All this effort creates a high energetic barrier the lipid undergoing flip–flop has to overcome, resulting in a slow and rare process when undertaken by a lone lipid.
The height of the energy barrier is dependent on both the properties of the translo- cating lipid and the surrounding lipid bilayer. The thickness of the bilayer affects the free energy profile of the flip–flopping lipid, the height of the energy barrier in- creasing along with the bilayer thickness. For instance, bilayers constituted of pure DLPC and DPPC were shown to have a fivefold difference in the height of the free energy barriers for flip–flops of their respective lipids (16 kJ/mol for DLPC and 80 kJ/mol for DPPC), even though the lengths of the lipid tails differ only by four car- bon atoms[104]. Lipid size also affects the rate of the flip–flop process, as shown by the ease with which cholesterol molecules are able to cross a lipid membrane, with free energy barrier heights generally below 30 kJ/mol[105]. The size and charge of the lipid headgroup are also important factors, with larger and more polar head- groups having greater difficulties penetrating the membrane surface [106]. Other relevant factors include the phase behavior of the bilayer[107]and the concentra- tion of membrane packing elements such as cholesterol[108].
There are several ways of reducing the free energy requirements of lipid flip–flop.
Defects in the bilayer may induce the formation of a small water pore, which creates a hydrophilic environment inside the bilayer for the translocating lipid to use. For protein-free membranes, thinner bilayers in particular have been shown to allow a water pore to form across the bilayer with relative ease, providing a convenient hy- drophilic path for the lipid headgroup to traverse[109]. These types of pore forma- tions have been shown to occur spontaneously without any external stimuli, albeit at a slow rate[110]. As spontaneous pore formation is the rate-limiting step when con- sidering lipid flip–flops in a defect-free membrane system, high enough rates of lipid flip–flop required for cellular sustainability are not possible to maintain by sponta- neous effects alone.
In the presence of external influences such as transmembrane peptides or various types of proteins, when the surface of the membrane is disturbed, these types of membrane defects occur more often, increasing the rate of pore formation[111].
Mechanical or electrical stress applied on the membrane surface also influences the rate with which water pores appear[112].
The energy requirements for lipid flip–flops can also be reduced by other means.
The following example is related to the modification of the membrane lipids. Oxida- tive stress is a chemical disturbance in the body where toxic reactive oxygen species are generated in excess resulting in extensive oxidative damage to lipids and proteins in a cellular environment[113]. This oxidative damage includes lipid peroxidation, often truncating the tail of a lipid with a double bond, fundamentally changing the characteristics of the lipid within the bilayer. Its effect on the free energy of lipid translocation has been studied, and the free energy barrier of POPS flip–flop in a pure POPC bilayer was found to reduce by 20 kJ/mol when 20 mol-% of the POPC molecules were substituted with oxidiced variants[114]. Such an energy reduction meant a 103– 104-fold increase in the flip–flop rates of POPS. Another recent study revealed a correlation between the decrease in the free energy of POPS flip–flop with increased concentrations of peroxidiced POPC molecules in a similar system[115].
Oxidative stress has implications in many neurogenerative diseases[116], or apop- tosis, where rapid PS translocation to the cellular surface is the trigger[117].