Scramblase is a
protein responsible for the translocation of
phospholipids between the two monolayers of a
lipid bilayer of a
cell membrane.[1][2][3] In humans, phospholipid scramblases (PLSCRs) constitute a family of five homologous proteins that are named as hPLSCR1–hPLSCR5. Scramblases are members of the general family of transmembrane lipid transporters known as
flippases. Scramblases are distinct from flippases and floppases. Scramblases, flippases, and floppases are three different types of enzymatic groups of phospholipid transportation enzymes.[4] The inner-leaflet, facing the inside of the cell, contains negatively charged amino-phospholipids and
phosphatidylethanolamine. The outer-leaflet, facing the outside environment, contains phosphatidylcholine and
sphingomyelin. Scramblase is an
enzyme, present in the cell membrane, that can transport (scramble) the negatively charged phospholipids from the inner-leaflet to the outer-leaflet, and vice versa.
Expression
Whereas hPLSCR1, -3, and -4 are expressed in a variety of tissues with few exceptions, expression of hPLSCR2 is restricted only to the
testis. hPLSCR4 is not expressed in peripheral blood
lymphocytes, whereas hPLSCR1 and -3 were not detected in the brain.[5] However, the functional significance of this differential gene expression is not yet understood. While the
gene and the
mRNA of hPLSCR5 provide evidence of its existence, the protein has yet to be described in the literature.
Structure
Scramblase proteins contain a region of conservation that possesses a 12-stranded
beta barrel surrounding a central
alpha helix.[6] This structure shows similarity to the
Tubby protein.
Enzyme activation
The enzymatic activity of scramblase depends on the
calcium concentration present inside the cell. The calcium concentration inside cells is, under normal conditions, very low; therefore, scramblase has a low activity under resting conditions. Phospholipid redistribution is triggered by increased cytosolic calcium and seems to be scramblase-dependent, resulting in a symmetric distribution of negatively charged phospholipids between both leaflets of the lipid bilayer. All scramblases contain an
EF hand-like Ca2+binding
domain that is probably responsible for the calcium activation of the enzyme. The activity of scramblase does not require energy, meaning that there is no contribution of
adenosine triphosphate in the process.
Scramblases are
proline-rich
proteins, possessing many cysteinyl sulfhydryl groups that are prone to modifications.
Oxidation,
nitrosylation, and blockage of these sulfhydryl groups produce an enhanced scramblase activity. Patients with
sickle cell disease exhibit a fraction of
erythrocytes with an aberrantly enhanced exposure of phosphatidyl serine on their surface. As the erythrocytes of these patients have an enhanced oxidative stress, it is probable that increased scramblase activity might play a role in the etiology of the disease. Furthermore, it is well recognized that both reactive oxygen species and intracellular Ca2+ fluxes affect mitochondria at the beginning of the apoptotic program. Sulfhydryl modification of PLSCR3 in mitochondria during apoptosis may be a key regulator initiating the intrinsic apoptotic pathways.
Nuclear localisation sequence
Phospholipid scramblase 1 (
PLSCR1), a lipid-binding protein that enters the
nucleus via the nonclassical
NLS (257)GKISKHWTGI(266). The structure of the nuclear localisation sequence of scramblase PLSCR1 complexed to
importin was determined using X-ray diffraction with a resolution of 2.20 Ångströms.[7] It is found in most mammals including humans. The import sequence lacks a continuous stretch of positively charged residues, and it is enriched in hydrophobic residues. Thus, Scramblase can transport negatively charged phospholipids from the inside of the cell to the outside of the cell. The importin structure is composed of many alpha helices that integrate the protein into membranes. The role of importin is to move proteins such as scramblase into the nucleus.
Biological roles
Mitochondrial membrane maintenance
Recent findings suggest that PLSCR3 is involved in regulation of biosynthesis of
cardiolipin in
mitochondria, and its overexpression in cultured cells resulted in increased
cardiolipin synthase[8][9] activity. As cardiolipin is synthesized in the luminal side of inner mitochondrial membrane, a major fraction of this newly synthesized pool of cardiolipin has to be translocated from the inner to the outer mitochondrial membrane. PLSCR3 has been proposed to be involved in this translocation from the inner to the outer membrane that is essential for maintaining the mitochondrial architecture, mass, and transmembrane potential.
Lipid metabolism
Recent findings suggest that PLSCR3 and, to a lesser degree, PLSCR1 are critical to the normal regulation of fat accumulation in mice. In addition to blood cells, PLSCR3 is expressed to a significantly higher level in fat and muscle cells, which are actively involved in fat
metabolism. PLSCR3 knockout mice showed an aberrant abdominal fat accumulation, glucose intolerance, insulin resistance, and dyslipidema as compared to controlled mice. Cultured fat cells from PLSCR3 knockout mice were engorged with neutral
lipids. Blood plasma of these mice showed elevated levels of non-high-density
lipoproteins,
cholesterol,
triglycerides, non-esterified
fatty acids, and
leptin, but low
adiponectin content. Abdominal fat accumulation with the formation of enlarged lipid engorged
adipocytes has emerged as the key risk factor for the onset of
type 2 diabetes,[10] which is often a manifestation of a broader underlying metabolic disorder termed as metabolic syndrome. Further studies on the regulation of lipid metabolism by PLSCRs are required to understand the risk for development of similar diseases in humans when PLSCR genes are mutated, leading to a defective expression and/or function of PLSCR proteins.
Thrombosis
Upon activation (in platelets) or injury (in erythrocytes, platelets, endothelium, and other cells), certain cells expose the
phospholipidphosphatidylserine on their surface and act as catalysts to induce the coagulation cascade. Surface exposure of phosphatidylserine is thought to be brought about by the activation of scramblases. Several enzyme complexes of blood coagulation cascade such as
tenase and
prothrombinase are activated by the cell surface exposure of the phosphatidylserine. However, the most studied member of the scramblase family PLSCR1 was shown to be defective in translocation of phospholipids when reconstituted into proteoliposomes in vitro. Although recent studies show that PLSCR1 is neither sufficient nor necessary for the phosphatidylserine externalization, the involvement of PLSCR1 in blood coagulation remains elusive, raising the question of additional membrane components in the externalization pathway. To date, no report is available on the involvement of any other identified member of PLSCRs in blood clotting.
Apoptosis
Apoptotic cell death is characterized by a
proteolyticcaspase cascade that emanates from either an extrinsic or an intrinsic pathway. The extrinsic pathway is initiated by membrane bound death receptors, leading to activation of
caspase 8, whereas the intrinsic pathway is triggered by DNA damaging drugs and UV radiation, leading to mitochondrial depolarization and subsequent activation of
caspase 9. PLSCRs are supposed to play an important role in both intrinsic and extrinsic apoptotic responses that are linked to each other via the activation of caspase 8. Activated caspase 8 causes the cleavage of the amino terminal portion of the cytosolic protein
Bid to generate t-Bid that is translocated into mitochondria during apoptosis. hPLSCR1 and its mitochondrial counterpart hPLSCR3 are phosphorylated by
PKCδ during PKC-δ-induced apoptosis. While the consequence of hPLSCR1 phosphorylation and its mechanism of action during cellular apoptotic response remain unclear, phosphorylated hPLSCR3 is thought to facilitate mitochondrial targeting of t-Bid that is an essential requirement in caspase 8-mediated apoptosis. The active t-Bid fragment is shown to localize to mitochondria through a positive interaction with cardiolipin. This activated t-Bid induces activation of
Bax and
Bak proteins to form
cytochrome c channels that facilitate the release of cytochrome c during apoptosis.
An early morphological event in both the extrinsic and the intrinsic apoptotic pathways is the surface exposure of the
phospholipidphosphatidylserine, about 96% of which normally reside in the cytosolic leaflet of the plasma membrane. Phosphatidylserine is translocated to the exoplasmic leaflet by the activation of scramblases, leading to pro-coagulant properties and providing a phagocytic signal to the
macrophages that engulf and clear the apoptotic cells. The involvement of other associated proteins aiding scrambling activity cannot be ruled out.
^Greenberg AS, McDaniel ML (June 2002). "Identifying the links between obesity, insulin resistance and beta-cell function: potential role of adipocyte-derived cytokines in the pathogenesis of type 2 diabetes". Eur. J. Clin. Invest. 32 (Suppl 3): 24–34.
doi:
10.1046/j.1365-2362.32.s3.4.x.
PMID12028372.
S2CID41305977.
Scramblase is a
protein responsible for the translocation of
phospholipids between the two monolayers of a
lipid bilayer of a
cell membrane.[1][2][3] In humans, phospholipid scramblases (PLSCRs) constitute a family of five homologous proteins that are named as hPLSCR1–hPLSCR5. Scramblases are members of the general family of transmembrane lipid transporters known as
flippases. Scramblases are distinct from flippases and floppases. Scramblases, flippases, and floppases are three different types of enzymatic groups of phospholipid transportation enzymes.[4] The inner-leaflet, facing the inside of the cell, contains negatively charged amino-phospholipids and
phosphatidylethanolamine. The outer-leaflet, facing the outside environment, contains phosphatidylcholine and
sphingomyelin. Scramblase is an
enzyme, present in the cell membrane, that can transport (scramble) the negatively charged phospholipids from the inner-leaflet to the outer-leaflet, and vice versa.
Expression
Whereas hPLSCR1, -3, and -4 are expressed in a variety of tissues with few exceptions, expression of hPLSCR2 is restricted only to the
testis. hPLSCR4 is not expressed in peripheral blood
lymphocytes, whereas hPLSCR1 and -3 were not detected in the brain.[5] However, the functional significance of this differential gene expression is not yet understood. While the
gene and the
mRNA of hPLSCR5 provide evidence of its existence, the protein has yet to be described in the literature.
Structure
Scramblase proteins contain a region of conservation that possesses a 12-stranded
beta barrel surrounding a central
alpha helix.[6] This structure shows similarity to the
Tubby protein.
Enzyme activation
The enzymatic activity of scramblase depends on the
calcium concentration present inside the cell. The calcium concentration inside cells is, under normal conditions, very low; therefore, scramblase has a low activity under resting conditions. Phospholipid redistribution is triggered by increased cytosolic calcium and seems to be scramblase-dependent, resulting in a symmetric distribution of negatively charged phospholipids between both leaflets of the lipid bilayer. All scramblases contain an
EF hand-like Ca2+binding
domain that is probably responsible for the calcium activation of the enzyme. The activity of scramblase does not require energy, meaning that there is no contribution of
adenosine triphosphate in the process.
Scramblases are
proline-rich
proteins, possessing many cysteinyl sulfhydryl groups that are prone to modifications.
Oxidation,
nitrosylation, and blockage of these sulfhydryl groups produce an enhanced scramblase activity. Patients with
sickle cell disease exhibit a fraction of
erythrocytes with an aberrantly enhanced exposure of phosphatidyl serine on their surface. As the erythrocytes of these patients have an enhanced oxidative stress, it is probable that increased scramblase activity might play a role in the etiology of the disease. Furthermore, it is well recognized that both reactive oxygen species and intracellular Ca2+ fluxes affect mitochondria at the beginning of the apoptotic program. Sulfhydryl modification of PLSCR3 in mitochondria during apoptosis may be a key regulator initiating the intrinsic apoptotic pathways.
Nuclear localisation sequence
Phospholipid scramblase 1 (
PLSCR1), a lipid-binding protein that enters the
nucleus via the nonclassical
NLS (257)GKISKHWTGI(266). The structure of the nuclear localisation sequence of scramblase PLSCR1 complexed to
importin was determined using X-ray diffraction with a resolution of 2.20 Ångströms.[7] It is found in most mammals including humans. The import sequence lacks a continuous stretch of positively charged residues, and it is enriched in hydrophobic residues. Thus, Scramblase can transport negatively charged phospholipids from the inside of the cell to the outside of the cell. The importin structure is composed of many alpha helices that integrate the protein into membranes. The role of importin is to move proteins such as scramblase into the nucleus.
Biological roles
Mitochondrial membrane maintenance
Recent findings suggest that PLSCR3 is involved in regulation of biosynthesis of
cardiolipin in
mitochondria, and its overexpression in cultured cells resulted in increased
cardiolipin synthase[8][9] activity. As cardiolipin is synthesized in the luminal side of inner mitochondrial membrane, a major fraction of this newly synthesized pool of cardiolipin has to be translocated from the inner to the outer mitochondrial membrane. PLSCR3 has been proposed to be involved in this translocation from the inner to the outer membrane that is essential for maintaining the mitochondrial architecture, mass, and transmembrane potential.
Lipid metabolism
Recent findings suggest that PLSCR3 and, to a lesser degree, PLSCR1 are critical to the normal regulation of fat accumulation in mice. In addition to blood cells, PLSCR3 is expressed to a significantly higher level in fat and muscle cells, which are actively involved in fat
metabolism. PLSCR3 knockout mice showed an aberrant abdominal fat accumulation, glucose intolerance, insulin resistance, and dyslipidema as compared to controlled mice. Cultured fat cells from PLSCR3 knockout mice were engorged with neutral
lipids. Blood plasma of these mice showed elevated levels of non-high-density
lipoproteins,
cholesterol,
triglycerides, non-esterified
fatty acids, and
leptin, but low
adiponectin content. Abdominal fat accumulation with the formation of enlarged lipid engorged
adipocytes has emerged as the key risk factor for the onset of
type 2 diabetes,[10] which is often a manifestation of a broader underlying metabolic disorder termed as metabolic syndrome. Further studies on the regulation of lipid metabolism by PLSCRs are required to understand the risk for development of similar diseases in humans when PLSCR genes are mutated, leading to a defective expression and/or function of PLSCR proteins.
Thrombosis
Upon activation (in platelets) or injury (in erythrocytes, platelets, endothelium, and other cells), certain cells expose the
phospholipidphosphatidylserine on their surface and act as catalysts to induce the coagulation cascade. Surface exposure of phosphatidylserine is thought to be brought about by the activation of scramblases. Several enzyme complexes of blood coagulation cascade such as
tenase and
prothrombinase are activated by the cell surface exposure of the phosphatidylserine. However, the most studied member of the scramblase family PLSCR1 was shown to be defective in translocation of phospholipids when reconstituted into proteoliposomes in vitro. Although recent studies show that PLSCR1 is neither sufficient nor necessary for the phosphatidylserine externalization, the involvement of PLSCR1 in blood coagulation remains elusive, raising the question of additional membrane components in the externalization pathway. To date, no report is available on the involvement of any other identified member of PLSCRs in blood clotting.
Apoptosis
Apoptotic cell death is characterized by a
proteolyticcaspase cascade that emanates from either an extrinsic or an intrinsic pathway. The extrinsic pathway is initiated by membrane bound death receptors, leading to activation of
caspase 8, whereas the intrinsic pathway is triggered by DNA damaging drugs and UV radiation, leading to mitochondrial depolarization and subsequent activation of
caspase 9. PLSCRs are supposed to play an important role in both intrinsic and extrinsic apoptotic responses that are linked to each other via the activation of caspase 8. Activated caspase 8 causes the cleavage of the amino terminal portion of the cytosolic protein
Bid to generate t-Bid that is translocated into mitochondria during apoptosis. hPLSCR1 and its mitochondrial counterpart hPLSCR3 are phosphorylated by
PKCδ during PKC-δ-induced apoptosis. While the consequence of hPLSCR1 phosphorylation and its mechanism of action during cellular apoptotic response remain unclear, phosphorylated hPLSCR3 is thought to facilitate mitochondrial targeting of t-Bid that is an essential requirement in caspase 8-mediated apoptosis. The active t-Bid fragment is shown to localize to mitochondria through a positive interaction with cardiolipin. This activated t-Bid induces activation of
Bax and
Bak proteins to form
cytochrome c channels that facilitate the release of cytochrome c during apoptosis.
An early morphological event in both the extrinsic and the intrinsic apoptotic pathways is the surface exposure of the
phospholipidphosphatidylserine, about 96% of which normally reside in the cytosolic leaflet of the plasma membrane. Phosphatidylserine is translocated to the exoplasmic leaflet by the activation of scramblases, leading to pro-coagulant properties and providing a phagocytic signal to the
macrophages that engulf and clear the apoptotic cells. The involvement of other associated proteins aiding scrambling activity cannot be ruled out.
^Greenberg AS, McDaniel ML (June 2002). "Identifying the links between obesity, insulin resistance and beta-cell function: potential role of adipocyte-derived cytokines in the pathogenesis of type 2 diabetes". Eur. J. Clin. Invest. 32 (Suppl 3): 24–34.
doi:
10.1046/j.1365-2362.32.s3.4.x.
PMID12028372.
S2CID41305977.