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Flippase and scramblase for phosphatidylserine exposure
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ScienceDirect Flippase and scramblase for phosphatidylserine exposure Shigekazu Nagata, Takaharu Sakuragi and Katsumori Segawa In various biological processes, phosphatidylserine (PtdSer) that is normally sequestered to the inner leaflet of the plasma membrane (PM) is exposed to the cell surface. When platelets are activated, they expose PtdSer to activate the blood-clotting factors. Cells undergoing apoptosis and senescent neutrophils expose PtdSer that is recognized as an ‘eat me’ signal by phagocytes for clearance. The PtdSer-exposure and its internalization are mediated by phospholipid scramblases and flippases, respectively. Both have recently been molecularly identified, and their functional mechanism and physiological roles are being elucidated. Address Laboratory of Biochemistry & Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan Corresponding author: Nagata, Shigekazu ([email protected])
Current Opinion in Immunology 2020, 62:31–38 This review comes from a themed issue on Innate immunity Edited by Frederic Geissmann and Jo Sun
https://doi.org/10.1016/j.coi.2019.11.009 0952-7915/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Eukaryotic plasma membranes (PMs) comprise a lipid bilayer of approximately 4 nm, and serve as barriers between the cell and its external environment. Lipids have a large diversity in their backbone [glycerophospholipids, sphingomyelins (SM), and cholesterol], acyl chain size, and number of double bonds [1]. Glycerophospholipids are further divided into phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), and phosphatidic acids. PMs are rich in PtdCho (43% of phospholipids), PtdEtn (21%), and SM (23%), whereas the content of PtdIns and PtdSer is approximately 7% and 4%, respectively [2]. Additionally, PMs, but not other intracellular membranes, contain high levels of cholesterol (50–80% of total phospholipids). www.sciencedirect.com
Glycerophospholipids, composed of hydrophilic head (phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidyl-serine and phosphatidyl-inositol), and hydrophobic tail groups (acyl chains), spontaneously form a lipid bilayer with the head groups facing outward and tail groups lining inward, due to these amphiphilic characteristics [3]. The asymmetrical distribution of phospholipids between PM outer and inner leaflets was noticed more than 25 years ago [4]. Soon after, several groups noticed that this asymmetry is disrupted in biological processes such as platelet activation and apoptosis [5,6]. To explain these processes, flippases that translocate PtdSer from the outer to inner leaflets and scramblases that scramble phospholipids between the lipid bilayers were postulated [7]. Both were recently identified [8], and we started to understand how and why phospholipids are translocated between lipid bilayer. Here, we will discuss the current understanding on flippases and scramblases in PtdSer-exposure.
Asymmetrical distribution of phospholipids The size of phospholipid headgroups as well as the length of acyl chains affects the shape of membranes [1]. Lipids with long and saturated fatty acids make membranes thicker and tighter, whereas unsaturated fatty acids prevent the tight packing. The size of PtdCho fatty acids is smaller (16 C at sn1, 18 C at sn2) than that of PtdEtn and PtdSer (18 C at sn1, 20 C at sn2) [9]. The majority of fatty acids at sn1 of PtdCho are saturated, whereas those of PtdEtn and PtdSer carry 4–6 unsaturated bonds, indicating that the membranes rich in PtdCho are tightly packed with low fluidity. The asymmetrical phospholipid distribution between the lipid bilayers was noticed in red blood cells, which lack intracellular membranes. Lipids in the external but not inner leaflets of PMs can be modified with chemical reagents or phospholipases, and the modified lipids are quantified in comparison with unmodified ones [4]. Using this method, 70–80% of SM and PtdCho were localized to the outer leaflet, whereas most of PtdSer and PtdEtn resided at the inner leaflets. Freeze-fracture replicalabelling immunoelectron microscopy with lipid-binding proteins recently showed that the asymmetrical distribution of phospholipids was more stringent: almost all PtdCho and SM were at their outer leaflets, whereas PtdSer, PtdEtn and PtdIns at the inner leaflets [10]. Compared with PMs, the phospholipid distribution in intracellular organelles (mitochondria, ER, and nuclei) is Current Opinion in Immunology 2020, 62:31–38
32 Innate immunity
difficult to determine. Yet, by expressing PtdSer-binding proteins in cells, one group localized PtdSer to the ER’s luminal leaflet [11], and suggested that PtdSer synthesized at the cytoplasmic leaflets flips to the luminal leaflet, and is transferred back to the inner leaflet when it moves to PMs. However, freeze–fracture replica labelling recently showed that PtdSer is much more abundant at the cytoplasmic than at the luminal leaflet of ER [12], suggesting that it is unnecessary for PtdSer to move twice to reach the PM, which may be a more likely model.
PtdCho were identified in the PMs and secretory vesicles [13,14]. The 115 120 kDa protein in chromaffin granules of bovine adrenal glands [15] and erythrocyte PMs [16] was characterized to have PtdSer-dependent ATPase activity, and assigned to aminophospholipid translocase or flippase [17]. Molecular cloning of cDNA encoding bovine chromaffin ATPase identified it as a member of the P4-subfamily of P-type ATPases with 10 transmembrane regions and two large cytoplasmic ATPase loops [18], most of which require CDC50A (TMEM30A) as a functional subunit for target localization [19–21] (Figure 1).
Flippases Tertiary structure of the P4-ATPase-CDC50A complex (yeast Drs2p-Cdc50p; and human ATP8A1-CDC50A)
Using spin-labeled or radioisotope-labeled phospholipids, ATP-dependent enzymes that translocate PtdSer, but not Figure 1
P4-ATPase
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Structure of P4-ATPase. (a) The structure of P4-ATPase is schematically shown. P4-ATPase comprised 10 transmembrane region (1–10), the Actuator (A) domain, the Nucleotide-binding (N) domain, and the Phosphorylation (P) domain in the cytoplasmic region. The P4-ATPase at plasma membrane is complexed with CDC50A carrying two transmembrane regions. CDC50A is required for localizing P4-ATPase to the plasma membrane and for its flippase activity. (b) The tertiary structure of yeast P4-ATPase (Drs2p-Cdc50p complex). The Cryo-EM density map (left) and cartoon representation of Drs2p-CDC50p complex (right). The A, N, and P domains are colored by yellow, red, and blue. The transmembrane regions are in tan. The CDC50A is colored by pink, while the unmodeled regions (lipid and detergent) are in orange. The images in (b) are from Timcenko et al. [23] with permission. Current Opinion in Immunology 2020, 62:31–38
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Flippases and scramblases for phosphatidylserine-exposure Nagata, Sakuragi and Segawa 33
was recently determined by cryoelectron microscopy and the molecular mechanism for lipid translocation was proposed [22,23]. The human P4-ATPase family comprises 14 members; eight of them are present at PMs, and three (ATP8A2, ATP11A and ATP11C) function as flippases at PMs. Unlike specific, spinal cord-specific, retina-specificATP8A2 that is specifically expressed in the brain spinal cord, retina, and testis [24–26], ATP11A and ATP11C are ubiquitously expressed and redundant in many cell types. Cells lacking both ATP11A and ATP11C genes have very little flippase activity at PMs, yet they do not expose PtdSer [27], suggesting that these flippases are dispensable for keeping the asymmetrical distribution of PtdSer in steady state. Treatment of the wild-type and ATP11A / ATP11C / cells with a Ca-ionophore causes the PtdSer-exposure due to Ca2+-dependent scramblase (see below). When the ionophore is removed, the PtdSer exposed on the cell surface is quickly internalized in the flippase-intact cells, but it
remained on the cell surface in ATP11A / ATP11C / cells [27], indicating that these flippases are required to quickly internalize scramblase-exposed PtdSer. Mouse fetal cardiomyocytes express only ATP11A, and ATP11A-null mice are embryonic lethal due to poor heart development [28]. Whereas, mouse B cell progenitors in the bone marrow express only ATP11C, and ATP11C-/mice suffer from B cell lymphopenia [29,30]. ATP11C / pre-B cells expose PtdSer, and are engulfed alive by macrophages [27]. When cells undergo apoptosis, they expose PtdSer within hours [31], whereas when platelets are activated, the PtdSer-exposure occurs within minutes [8]. ATP11A and ATP11C are cleaved and inactivated by caspase 3 during apoptosis, while their ATPase activity is inhibited by a high concentration of Ca2+ [24,32]. However, flippase inactivation is insufficient to expose PtdSer quickly in apoptotic cells or activated platelets. Once the asymmetrical
Figure 2
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Structure of TMEM16F. (a) The Cryo-EM map (left) and the cartoon representation of the Ca2+-bound form of mouse TMEM16 F. The 10 transmembrane regions (a1-a10) are indicated. (b) A proposed mechanism for the lipid scrambling by TMEM16F. In the absence of Ca2+, the lipid conductive cavity formed by the transmembrane region of 3–7 (a3–a7) is closed. When two Ca2+ (blue circle) bound to the molecule, the cavity is opened for passing phospholipids that are consisted of hydrophilic head groups (red) and hydrophobic acyl chains. The scrambling domain (SCRD) critical for passing phospholipids [44,68] is represented in pink. The images are from Alvadia et al. [39]. www.sciencedirect.com
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Figure 3
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Steady State CDC50A TMEM16F (homodimer)
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The phosphatidylserine-exposure regulated by flippases and scramblases. In steady state (a), ATP11A and ATP11C of the P4-ATPase family together with CDC50A flip or actively translocate PtdSer and PtdEtn from the outer to inner leaflets of the plasma membranes. The two scramblases, a TMEM16F homodimer and an Xkr8 and Basigin (or Neuroplastin) heterodimer, are dormant. (b) When the intracellular Ca2+ concentration increases, Ca2+ inhibits the flippase activity of ATP11A and ATP11C. It binds TMEM16F and causes its conformational change to function as a scramblase. This process is responsible for the PtdSer-exposure and release of microparticles in the activated platelets as well as the PtdSer-exposure and deposition of hydroxyapatite in mature osteoblasts during skeletogenesis. (c) When cells undergo apoptosis, caspase
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distribution of phospholipids is established, it takes days to destroy it [33], because the translocation of the hydrophilic head of phospholipids through hydrophobic lipid layers does not occur easily. Thus, a scramblase that quickly scrambles phospholipids between lipid bilayer is necessary to expose PtdSer swiftly.
TMEM16F plays a role in releasing matrix vesicles containing hydroxylapatite from osteoblasts [50] and in shedding ectosomes from activated T lymphocytes [51]. This is probably because TMEM16F-mediated scrambling of phospholipids changes the membrane curvature, causing PM scission and vesicular release [52].
Scramblases TMEM16
Members of two families (TMEM16 and XKR) of membrane proteins with ten transmembrane regions can act as phospholipid scramblases. When we identified TMEM16 F as a Ca2+-dependent scramblase [34], there was a possibility that the scramblase activity was mediated by other molecules associated with TMEM16F [35]. By reconstituting in liposomes, fungus TMEM16F orthologues were shown to have the scramblase activity [36,37]. Furthermore, Watanabe et al. [38] recently established a microarray system in which the fluorescence-labeled PtdSer is present only in the upper layer of the lipid bilayer formed over each well, and showed that homogeneous TMEM16F has Ca2+-dependent scramblase activity at single molecule level. The tertiary structure of TMEM16 was at first determined for the fungus orthologue, and then for mouse TMEM16F in Ca2+-bound or unbound forms [36,39–41] (Figure 2). TMEM16F forms a homodimer with a ‘subunit cavity’ that contains a few polar residues, and causes lipid bilayer thinning at a specific area. Binding of Ca2+ to TMEM16F induces its conformational change, opens the cavity to pass phospholipids. This model is called a ‘stepping stone’ or ‘credit card’ model [42,43]. Five human TMEM16F members (16C, 16D, 16F, 16G and 16J) are present at the PMs, carry the conserved charged amino acids in the transmembrane regions [44], and have the scramblase activity. Even some members in ER (TMEM16E and 16K) carry the conserved charged residues [44], and the scrambling activity of TMEM16K was shown in a reconstitution system [45], suggesting that it may work as a Ca2+-dependent scramblase in ER [12]. TMEM16F and TMEM16K are ubiquitously expressed in various cells, whereas others are expressed in specific tissues [46]. TMEM16F is the only Ca2+-dependent scramblase expressed at the platelet’s PM [47]. We and others showed that patients of Scott syndrome, a bleeding disorder in which platelets cannot expose PtdSer, carry recessive mutation in TMEM16F [34,48,49]. The platelet-specific deletion of TMEM16F in mouse caused defects in the activation-induced PtdSer-exposure and microparticle shedding, leading to reduced thrombin production [47].
XKR
A cell line established from TMEM16F / fetal thymocytes does not expose PtdSer in response to a Ca2+-ionophore, but exposes PtdSer upon apoptotic stimuli [46]. In contrast, Xkr8 / cells respond to a Ca2+-ionophore as efficiently as the wild-type cells, but do not respond to apoptotic stimuli [53], indicating that Ca2+- and apoptosis-induced PtdSerexposures are mediated by two different scramblase systems (Figure 3). Xkr8 is a ubiquitously expressed member of the XK-family [53]. It is complexed with Basigin (Bsg) or Neuroplastin (NPTN), type I membrane proteins of Immunoglobulinsuperfamily that serve as chaperones to localize Xkr8 to PMs [54]. Xkr8 carries a well-conserved caspase-recognition site in its C-terminal tail. Its cleavage by caspase during apoptosis causes dimerization of the Xkr8-Bsg/NPTNcomplex into a hetero-tetramer to function as a scramblase [54]. As the flippases (ATP11A and ATP11C) are destroyed by caspase-cleavage, once PtdSer is exposed to the cell surface by Xkr8, it does not return to the inner leaflet, and serves as an ‘eat me’ signal. Neutrophils have a very short life-time (12 hours) in the blood stream [55]. Senescent neutrophils expose PtdSer, and are engulfed by macrophages in the liver, spleen, and bone marrow [56]. PtdSer exposure in senescent neutrophils depends on the caspaseactivated Xkr8 [57], indicating that cellular senescence, at least in neutrophils, proceeds via apoptosis. Approximately 109 neutrophils per kg of bodyweight are released into the blood stream per day, and the same number of cells are cleared by macrophages [56]. Defects in PtdSer-dependent engulfment of dead cells causes Systemic Lupus Erythematosus-type autoimmune diseases in pro-autoimmune mouse strains [58–60]. Similarly, Xkr8 / mice that accumulate unengulfed apoptotic cells and senescent neutrophils develop autoimmunity in MRL mouse strain [57]. Among the 9 Xkr family members, Xkr4 and Xkr9 are expressed in the brain and intestines, respectively, and have the caspase-activated scramblase activity [61]. Why these tissues express an additional caspase-dependent scramblase is puzzling, and whether they have a specific role in the brain and intestines remains to be studied.
(Figure 3 Legend Continued) 3 cleaves and inactivates the flippases, while it cleaves the Xkr8-BSG heterodimer to activate. The activated Xkr8-BSG complex undergoes multimerization to work as a scramblase. The cleavage by caspase is an irreversible process, and the PtdSer irreversibly exposed to the cell surface serves as an ‘eat me’ signal. (d) In unidentified processes, a specific kinase(s) inhibits the flippase activity of ATP11A and ATP11C by phosphorylation, while the Xkr8 scramblase activity is activated by phosphorylation, leading to the PtdSer-exposure. www.sciencedirect.com
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Table 1 Biological processes that cause the PtdSer-exposure. Biological processes that cause the PtdSer-exposure are listed. Scramblases that are responsible or possibly responsible for the PtdSer-exposure are shown with its stimulator Cells or organisms Apoptotic cells Senescent neutrophils Activated platelets (release of microparticles) Activated osteoblasts (release of hydroxyapatite) Senescent red blood cells Necroptotic cells (Cells undergoing pyroptosis or necroptosis) Activated lymphocytes Activated mast cells (releasing histamine) Some tumor cells and tumor-associated endothelial cells Fusing osteoclast Capacitated sperm Pyrenocytes (nuclei released from erythroblasts) Enveloped virus Milk Fat Globules
Besides caspase-mediated cleavage, Xkr8 can be activated by phosphorylation [62] (Figure 3). When IL-3-dependent Ba/F3 pro-B cells were transformed with mouse Xkr8 and Bsg, the transformants exposed PtdSer in a kinasedependent manner. Mass-spectrometry analysis showed that mouse Xkr8 is phosphorylated at serine or threonine residues downstream of the caspase recognition site of its C-terminal tail region, and activated. The amino acid sequence (approximately 40 amino acids) of this tail region is well conserved among mammals. We speculate that this region serves as an inhibitory domain by masking the phospholipid-entry site in Xkr8 or by preventing its dimerization. Caspases simply remove the domain, while the phosphorylation may prevent its target interaction. In contrast with Ba/F3 cells, the overexpression of Xkr8 in mouse WR19 L or human PLB cells did not constitutively activate Xkr8. It is likely that a specific kinase(s) expressed only in certain type of cells is responsible for this activation. In any case, the reversible regulation of Xkr8 scrambling activity by phosphorylation indicates that this system is involved in various biological processes that expose PtdSer.
scramblase XKR8 XKR8 TMEM16F TMEM16F TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or
XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8?
effector caspase caspase Ca2+ Ca2+ Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase?
nuclei), milk fat globules released by mammary glands, and enveloped viruses). Asymmetrical distribution and scrambling of phospholipids are observed in intracellular membranes, too, but its physiological roles are unknown. The P4-ATPases, TMEM16 and Xkr form big families (9–14 members) [63] that can be regulated not only by Ca2+ and caspases, but also by phosphorylation [62,65]. Defects or mutations in their family members cause various human diseases [63,66,67]. Therefore, elucidation of their molecular mechanisms would contribute to the understanding of these diseases.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements We thank all members in our laboratory. The work in our laboratory was supported by Japan Science Promotion Society (JSPS) 15H05785 (to S.N.), and Japan Science Technology (JST) JPMJCR14M4 (to S.N.).
Perspectives With the tertiary structure of some of P4-ATPases and TMEM16F scramblases [22,23,36,39,40], we now start to understand how flippases translocate PtdSer in an ATPdependent manner, and how the scramblases scramble phospholipids between lipid bilayers. We also understand PtdSer exposure in activated platelets, in apoptotic cells, and senescent neutrophils [63,64] (Table 1). Nevertheless, many unexplored problems remain. For example, the plasma membrane flippases are dispensable to maintain the asymmetrical distribution of phospholipids in steady state [27], of which the mechanism is unknown. The PtdSer-exposure occurs in many processes (i.e., activated mast cells, fusing osteoclasts, activated lymphocytes, capacitated sperm, some cancer cells, senescent red blood cells, erythroblast-released pyrenocytes (PM-covered Current Opinion in Immunology 2020, 62:31–38
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ScienceDirect Flippase and scramblase for phosphatidylserine exposure Shigekazu Nagata, Takaharu Sakuragi and Katsumori Segawa In various biological processes, phosphatidylserine (PtdSer) that is normally sequestered to the inner leaflet of the plasma membrane (PM) is exposed to the cell surface. When platelets are activated, they expose PtdSer to activate the blood-clotting factors. Cells undergoing apoptosis and senescent neutrophils expose PtdSer that is recognized as an ‘eat me’ signal by phagocytes for clearance. The PtdSer-exposure and its internalization are mediated by phospholipid scramblases and flippases, respectively. Both have recently been molecularly identified, and their functional mechanism and physiological roles are being elucidated. Address Laboratory of Biochemistry & Immunology, World Premier International Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan Corresponding author: Nagata, Shigekazu ([email protected])
Current Opinion in Immunology 2020, 62:31–38 This review comes from a themed issue on Innate immunity Edited by Frederic Geissmann and Jo Sun
https://doi.org/10.1016/j.coi.2019.11.009 0952-7915/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Eukaryotic plasma membranes (PMs) comprise a lipid bilayer of approximately 4 nm, and serve as barriers between the cell and its external environment. Lipids have a large diversity in their backbone [glycerophospholipids, sphingomyelins (SM), and cholesterol], acyl chain size, and number of double bonds [1]. Glycerophospholipids are further divided into phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), phosphatidylinositol (PtdIns), and phosphatidic acids. PMs are rich in PtdCho (43% of phospholipids), PtdEtn (21%), and SM (23%), whereas the content of PtdIns and PtdSer is approximately 7% and 4%, respectively [2]. Additionally, PMs, but not other intracellular membranes, contain high levels of cholesterol (50–80% of total phospholipids). www.sciencedirect.com
Glycerophospholipids, composed of hydrophilic head (phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidyl-serine and phosphatidyl-inositol), and hydrophobic tail groups (acyl chains), spontaneously form a lipid bilayer with the head groups facing outward and tail groups lining inward, due to these amphiphilic characteristics [3]. The asymmetrical distribution of phospholipids between PM outer and inner leaflets was noticed more than 25 years ago [4]. Soon after, several groups noticed that this asymmetry is disrupted in biological processes such as platelet activation and apoptosis [5,6]. To explain these processes, flippases that translocate PtdSer from the outer to inner leaflets and scramblases that scramble phospholipids between the lipid bilayers were postulated [7]. Both were recently identified [8], and we started to understand how and why phospholipids are translocated between lipid bilayer. Here, we will discuss the current understanding on flippases and scramblases in PtdSer-exposure.
Asymmetrical distribution of phospholipids The size of phospholipid headgroups as well as the length of acyl chains affects the shape of membranes [1]. Lipids with long and saturated fatty acids make membranes thicker and tighter, whereas unsaturated fatty acids prevent the tight packing. The size of PtdCho fatty acids is smaller (16 C at sn1, 18 C at sn2) than that of PtdEtn and PtdSer (18 C at sn1, 20 C at sn2) [9]. The majority of fatty acids at sn1 of PtdCho are saturated, whereas those of PtdEtn and PtdSer carry 4–6 unsaturated bonds, indicating that the membranes rich in PtdCho are tightly packed with low fluidity. The asymmetrical phospholipid distribution between the lipid bilayers was noticed in red blood cells, which lack intracellular membranes. Lipids in the external but not inner leaflets of PMs can be modified with chemical reagents or phospholipases, and the modified lipids are quantified in comparison with unmodified ones [4]. Using this method, 70–80% of SM and PtdCho were localized to the outer leaflet, whereas most of PtdSer and PtdEtn resided at the inner leaflets. Freeze-fracture replicalabelling immunoelectron microscopy with lipid-binding proteins recently showed that the asymmetrical distribution of phospholipids was more stringent: almost all PtdCho and SM were at their outer leaflets, whereas PtdSer, PtdEtn and PtdIns at the inner leaflets [10]. Compared with PMs, the phospholipid distribution in intracellular organelles (mitochondria, ER, and nuclei) is Current Opinion in Immunology 2020, 62:31–38
32 Innate immunity
difficult to determine. Yet, by expressing PtdSer-binding proteins in cells, one group localized PtdSer to the ER’s luminal leaflet [11], and suggested that PtdSer synthesized at the cytoplasmic leaflets flips to the luminal leaflet, and is transferred back to the inner leaflet when it moves to PMs. However, freeze–fracture replica labelling recently showed that PtdSer is much more abundant at the cytoplasmic than at the luminal leaflet of ER [12], suggesting that it is unnecessary for PtdSer to move twice to reach the PM, which may be a more likely model.
PtdCho were identified in the PMs and secretory vesicles [13,14]. The 115 120 kDa protein in chromaffin granules of bovine adrenal glands [15] and erythrocyte PMs [16] was characterized to have PtdSer-dependent ATPase activity, and assigned to aminophospholipid translocase or flippase [17]. Molecular cloning of cDNA encoding bovine chromaffin ATPase identified it as a member of the P4-subfamily of P-type ATPases with 10 transmembrane regions and two large cytoplasmic ATPase loops [18], most of which require CDC50A (TMEM30A) as a functional subunit for target localization [19–21] (Figure 1).
Flippases Tertiary structure of the P4-ATPase-CDC50A complex (yeast Drs2p-Cdc50p; and human ATP8A1-CDC50A)
Using spin-labeled or radioisotope-labeled phospholipids, ATP-dependent enzymes that translocate PtdSer, but not Figure 1
P4-ATPase
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Structure of P4-ATPase. (a) The structure of P4-ATPase is schematically shown. P4-ATPase comprised 10 transmembrane region (1–10), the Actuator (A) domain, the Nucleotide-binding (N) domain, and the Phosphorylation (P) domain in the cytoplasmic region. The P4-ATPase at plasma membrane is complexed with CDC50A carrying two transmembrane regions. CDC50A is required for localizing P4-ATPase to the plasma membrane and for its flippase activity. (b) The tertiary structure of yeast P4-ATPase (Drs2p-Cdc50p complex). The Cryo-EM density map (left) and cartoon representation of Drs2p-CDC50p complex (right). The A, N, and P domains are colored by yellow, red, and blue. The transmembrane regions are in tan. The CDC50A is colored by pink, while the unmodeled regions (lipid and detergent) are in orange. The images in (b) are from Timcenko et al. [23] with permission. Current Opinion in Immunology 2020, 62:31–38
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Flippases and scramblases for phosphatidylserine-exposure Nagata, Sakuragi and Segawa 33
was recently determined by cryoelectron microscopy and the molecular mechanism for lipid translocation was proposed [22,23]. The human P4-ATPase family comprises 14 members; eight of them are present at PMs, and three (ATP8A2, ATP11A and ATP11C) function as flippases at PMs. Unlike specific, spinal cord-specific, retina-specificATP8A2 that is specifically expressed in the brain spinal cord, retina, and testis [24–26], ATP11A and ATP11C are ubiquitously expressed and redundant in many cell types. Cells lacking both ATP11A and ATP11C genes have very little flippase activity at PMs, yet they do not expose PtdSer [27], suggesting that these flippases are dispensable for keeping the asymmetrical distribution of PtdSer in steady state. Treatment of the wild-type and ATP11A / ATP11C / cells with a Ca-ionophore causes the PtdSer-exposure due to Ca2+-dependent scramblase (see below). When the ionophore is removed, the PtdSer exposed on the cell surface is quickly internalized in the flippase-intact cells, but it
remained on the cell surface in ATP11A / ATP11C / cells [27], indicating that these flippases are required to quickly internalize scramblase-exposed PtdSer. Mouse fetal cardiomyocytes express only ATP11A, and ATP11A-null mice are embryonic lethal due to poor heart development [28]. Whereas, mouse B cell progenitors in the bone marrow express only ATP11C, and ATP11C-/mice suffer from B cell lymphopenia [29,30]. ATP11C / pre-B cells expose PtdSer, and are engulfed alive by macrophages [27]. When cells undergo apoptosis, they expose PtdSer within hours [31], whereas when platelets are activated, the PtdSer-exposure occurs within minutes [8]. ATP11A and ATP11C are cleaved and inactivated by caspase 3 during apoptosis, while their ATPase activity is inhibited by a high concentration of Ca2+ [24,32]. However, flippase inactivation is insufficient to expose PtdSer quickly in apoptotic cells or activated platelets. Once the asymmetrical
Figure 2
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Structure of TMEM16F. (a) The Cryo-EM map (left) and the cartoon representation of the Ca2+-bound form of mouse TMEM16 F. The 10 transmembrane regions (a1-a10) are indicated. (b) A proposed mechanism for the lipid scrambling by TMEM16F. In the absence of Ca2+, the lipid conductive cavity formed by the transmembrane region of 3–7 (a3–a7) is closed. When two Ca2+ (blue circle) bound to the molecule, the cavity is opened for passing phospholipids that are consisted of hydrophilic head groups (red) and hydrophobic acyl chains. The scrambling domain (SCRD) critical for passing phospholipids [44,68] is represented in pink. The images are from Alvadia et al. [39]. www.sciencedirect.com
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Figure 3
(a)
Steady State CDC50A TMEM16F (homodimer)
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The phosphatidylserine-exposure regulated by flippases and scramblases. In steady state (a), ATP11A and ATP11C of the P4-ATPase family together with CDC50A flip or actively translocate PtdSer and PtdEtn from the outer to inner leaflets of the plasma membranes. The two scramblases, a TMEM16F homodimer and an Xkr8 and Basigin (or Neuroplastin) heterodimer, are dormant. (b) When the intracellular Ca2+ concentration increases, Ca2+ inhibits the flippase activity of ATP11A and ATP11C. It binds TMEM16F and causes its conformational change to function as a scramblase. This process is responsible for the PtdSer-exposure and release of microparticles in the activated platelets as well as the PtdSer-exposure and deposition of hydroxyapatite in mature osteoblasts during skeletogenesis. (c) When cells undergo apoptosis, caspase
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distribution of phospholipids is established, it takes days to destroy it [33], because the translocation of the hydrophilic head of phospholipids through hydrophobic lipid layers does not occur easily. Thus, a scramblase that quickly scrambles phospholipids between lipid bilayer is necessary to expose PtdSer swiftly.
TMEM16F plays a role in releasing matrix vesicles containing hydroxylapatite from osteoblasts [50] and in shedding ectosomes from activated T lymphocytes [51]. This is probably because TMEM16F-mediated scrambling of phospholipids changes the membrane curvature, causing PM scission and vesicular release [52].
Scramblases TMEM16
Members of two families (TMEM16 and XKR) of membrane proteins with ten transmembrane regions can act as phospholipid scramblases. When we identified TMEM16 F as a Ca2+-dependent scramblase [34], there was a possibility that the scramblase activity was mediated by other molecules associated with TMEM16F [35]. By reconstituting in liposomes, fungus TMEM16F orthologues were shown to have the scramblase activity [36,37]. Furthermore, Watanabe et al. [38] recently established a microarray system in which the fluorescence-labeled PtdSer is present only in the upper layer of the lipid bilayer formed over each well, and showed that homogeneous TMEM16F has Ca2+-dependent scramblase activity at single molecule level. The tertiary structure of TMEM16 was at first determined for the fungus orthologue, and then for mouse TMEM16F in Ca2+-bound or unbound forms [36,39–41] (Figure 2). TMEM16F forms a homodimer with a ‘subunit cavity’ that contains a few polar residues, and causes lipid bilayer thinning at a specific area. Binding of Ca2+ to TMEM16F induces its conformational change, opens the cavity to pass phospholipids. This model is called a ‘stepping stone’ or ‘credit card’ model [42,43]. Five human TMEM16F members (16C, 16D, 16F, 16G and 16J) are present at the PMs, carry the conserved charged amino acids in the transmembrane regions [44], and have the scramblase activity. Even some members in ER (TMEM16E and 16K) carry the conserved charged residues [44], and the scrambling activity of TMEM16K was shown in a reconstitution system [45], suggesting that it may work as a Ca2+-dependent scramblase in ER [12]. TMEM16F and TMEM16K are ubiquitously expressed in various cells, whereas others are expressed in specific tissues [46]. TMEM16F is the only Ca2+-dependent scramblase expressed at the platelet’s PM [47]. We and others showed that patients of Scott syndrome, a bleeding disorder in which platelets cannot expose PtdSer, carry recessive mutation in TMEM16F [34,48,49]. The platelet-specific deletion of TMEM16F in mouse caused defects in the activation-induced PtdSer-exposure and microparticle shedding, leading to reduced thrombin production [47].
XKR
A cell line established from TMEM16F / fetal thymocytes does not expose PtdSer in response to a Ca2+-ionophore, but exposes PtdSer upon apoptotic stimuli [46]. In contrast, Xkr8 / cells respond to a Ca2+-ionophore as efficiently as the wild-type cells, but do not respond to apoptotic stimuli [53], indicating that Ca2+- and apoptosis-induced PtdSerexposures are mediated by two different scramblase systems (Figure 3). Xkr8 is a ubiquitously expressed member of the XK-family [53]. It is complexed with Basigin (Bsg) or Neuroplastin (NPTN), type I membrane proteins of Immunoglobulinsuperfamily that serve as chaperones to localize Xkr8 to PMs [54]. Xkr8 carries a well-conserved caspase-recognition site in its C-terminal tail. Its cleavage by caspase during apoptosis causes dimerization of the Xkr8-Bsg/NPTNcomplex into a hetero-tetramer to function as a scramblase [54]. As the flippases (ATP11A and ATP11C) are destroyed by caspase-cleavage, once PtdSer is exposed to the cell surface by Xkr8, it does not return to the inner leaflet, and serves as an ‘eat me’ signal. Neutrophils have a very short life-time (12 hours) in the blood stream [55]. Senescent neutrophils expose PtdSer, and are engulfed by macrophages in the liver, spleen, and bone marrow [56]. PtdSer exposure in senescent neutrophils depends on the caspaseactivated Xkr8 [57], indicating that cellular senescence, at least in neutrophils, proceeds via apoptosis. Approximately 109 neutrophils per kg of bodyweight are released into the blood stream per day, and the same number of cells are cleared by macrophages [56]. Defects in PtdSer-dependent engulfment of dead cells causes Systemic Lupus Erythematosus-type autoimmune diseases in pro-autoimmune mouse strains [58–60]. Similarly, Xkr8 / mice that accumulate unengulfed apoptotic cells and senescent neutrophils develop autoimmunity in MRL mouse strain [57]. Among the 9 Xkr family members, Xkr4 and Xkr9 are expressed in the brain and intestines, respectively, and have the caspase-activated scramblase activity [61]. Why these tissues express an additional caspase-dependent scramblase is puzzling, and whether they have a specific role in the brain and intestines remains to be studied.
(Figure 3 Legend Continued) 3 cleaves and inactivates the flippases, while it cleaves the Xkr8-BSG heterodimer to activate. The activated Xkr8-BSG complex undergoes multimerization to work as a scramblase. The cleavage by caspase is an irreversible process, and the PtdSer irreversibly exposed to the cell surface serves as an ‘eat me’ signal. (d) In unidentified processes, a specific kinase(s) inhibits the flippase activity of ATP11A and ATP11C by phosphorylation, while the Xkr8 scramblase activity is activated by phosphorylation, leading to the PtdSer-exposure. www.sciencedirect.com
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Table 1 Biological processes that cause the PtdSer-exposure. Biological processes that cause the PtdSer-exposure are listed. Scramblases that are responsible or possibly responsible for the PtdSer-exposure are shown with its stimulator Cells or organisms Apoptotic cells Senescent neutrophils Activated platelets (release of microparticles) Activated osteoblasts (release of hydroxyapatite) Senescent red blood cells Necroptotic cells (Cells undergoing pyroptosis or necroptosis) Activated lymphocytes Activated mast cells (releasing histamine) Some tumor cells and tumor-associated endothelial cells Fusing osteoclast Capacitated sperm Pyrenocytes (nuclei released from erythroblasts) Enveloped virus Milk Fat Globules
Besides caspase-mediated cleavage, Xkr8 can be activated by phosphorylation [62] (Figure 3). When IL-3-dependent Ba/F3 pro-B cells were transformed with mouse Xkr8 and Bsg, the transformants exposed PtdSer in a kinasedependent manner. Mass-spectrometry analysis showed that mouse Xkr8 is phosphorylated at serine or threonine residues downstream of the caspase recognition site of its C-terminal tail region, and activated. The amino acid sequence (approximately 40 amino acids) of this tail region is well conserved among mammals. We speculate that this region serves as an inhibitory domain by masking the phospholipid-entry site in Xkr8 or by preventing its dimerization. Caspases simply remove the domain, while the phosphorylation may prevent its target interaction. In contrast with Ba/F3 cells, the overexpression of Xkr8 in mouse WR19 L or human PLB cells did not constitutively activate Xkr8. It is likely that a specific kinase(s) expressed only in certain type of cells is responsible for this activation. In any case, the reversible regulation of Xkr8 scrambling activity by phosphorylation indicates that this system is involved in various biological processes that expose PtdSer.
scramblase XKR8 XKR8 TMEM16F TMEM16F TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or TMEM16F or
XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8? XKR8?
effector caspase caspase Ca2+ Ca2+ Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase? Ca2+ or Kinase?
nuclei), milk fat globules released by mammary glands, and enveloped viruses). Asymmetrical distribution and scrambling of phospholipids are observed in intracellular membranes, too, but its physiological roles are unknown. The P4-ATPases, TMEM16 and Xkr form big families (9–14 members) [63] that can be regulated not only by Ca2+ and caspases, but also by phosphorylation [62,65]. Defects or mutations in their family members cause various human diseases [63,66,67]. Therefore, elucidation of their molecular mechanisms would contribute to the understanding of these diseases.
Declaration of interests
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements We thank all members in our laboratory. The work in our laboratory was supported by Japan Science Promotion Society (JSPS) 15H05785 (to S.N.), and Japan Science Technology (JST) JPMJCR14M4 (to S.N.).
Perspectives With the tertiary structure of some of P4-ATPases and TMEM16F scramblases [22,23,36,39,40], we now start to understand how flippases translocate PtdSer in an ATPdependent manner, and how the scramblases scramble phospholipids between lipid bilayers. We also understand PtdSer exposure in activated platelets, in apoptotic cells, and senescent neutrophils [63,64] (Table 1). Nevertheless, many unexplored problems remain. For example, the plasma membrane flippases are dispensable to maintain the asymmetrical distribution of phospholipids in steady state [27], of which the mechanism is unknown. The PtdSer-exposure occurs in many processes (i.e., activated mast cells, fusing osteoclasts, activated lymphocytes, capacitated sperm, some cancer cells, senescent red blood cells, erythroblast-released pyrenocytes (PM-covered Current Opinion in Immunology 2020, 62:31–38
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