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Phospholipase D (, ''lipophosphodiesterase II'', ''lecithinase D'', ''choline phosphatase'') (PLD) is an
enzyme Enzymes () are proteins that act as biological catalysts (biocatalysts). Catalysts accelerate chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates int ...
of the
phospholipase Phospholipase cleavage sites. An enzyme that displays both PLA1 and PLA2 activities is called a '' Phospholipase B''. A phospholipase is an enzyme that hydrolyzes phospholipids into fatty acid fatty acids have perfectly straight chain structure. ...
superfamily. Phospholipases occur widely, and can be found in a wide range of organisms, including bacteria, yeast, plants, animals, and viruses. Phospholipase D's principal substrate is
phosphatidylcholine Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup. They are a major component of biological membranes and can be easily obtained from a variety of readily available sources, such as egg yolk or soybeans, ...
, which it
hydrolyzes
hydrolyzes
to produce the
signal In signal processing Signal processing is an electrical engineering subfield that focuses on analysing, modifying, and synthesizing signals such as audio signal processing, sound, image processing, images, and scientific measurements. Sig ...
molecule phosphatidic acid (PA), and
soluble Solubility is the property of a solid, liquid or gaseous chemical substance called ''solution, solute'' to dissolve in a solid, liquid or gaseous solvent. The solubility of a substance fundamentally depends on the Physical property, physical an ...
choline Choline is an essential nutrient for humans and many other animals. Choline occurs as a cation An ion () is a particle, atom or molecule with a net electric charge, electrical charge. The charge of the electron is considered negative by ...
. Plants contain numerous genes that encode various PLD isoenzymes, with
molecular weights The molecular mass (''m'') is the mass of a given molecule: it is measured in daltons (Da or u). Different molecules of the same compound may have different molecular masses because they contain different isotope Isotopes are variants of a par ...
ranging from 90 to 125 kDa. Mammalian cells encode two isoforms of phospholipase D: PLD1 and PLD2. Phospholipase D is an important player in many
physiological Physiology (; ) is the scientific study of functions and mechanisms in a living system. As a sub-discipline of biology Biology is the natural science that studies life and living organisms, including their anatomy, physical structure, B ...
processes, including membrane trafficking,
cytoskeletal Image:FluorescentCells.jpg, 300px, The eukaryotic cytoskeleton. Actin filaments are shown in red, and microtubules composed of beta tubulin are in green. The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in t ...
reorganization,
receptor-mediated endocytosis Image:Itrafig2.jpg, 400px, Mechanism of clathrin-dependent endocytosis Receptor-mediated endocytosis (RME), also called clathrin-mediated endocytosis, is a process by which cells absorb metabolites, hormones, proteins ā€“ and in some cases viruses ā ...
, exocytosis, and cell migration. Through these processes, it has been further implicated in the pathophysiology of multiple diseases: in particular the progression of Parkinson's disease, Parkinson's and Alzheimer's disease, Alzheimer's, as well as various cancers. PLD may also help set the threshold for sensitivity to anesthetia and mechanical force.


Discovery

PLD-type Enzyme assay, activity was first reported in 1947 by Donald J. Hanahan and I.L. Chaikoff. It was not until 1975, however, that the hydrolysis, hydrolytic mechanism of action was elucidated in mammals, mammalian cells. Plant isoforms of PLD were first Protein purification, purified from cabbage and castor bean; PLDĪ± was ultimately cloning, cloned and characterized from a variety of plants, including rice, corn, and tomato. Plant PLDs have been cloned in three isoforms: PLDĪ±, PLDĪ², and PLDĪ³. More than half a century of biochemical studies have implicated phospholipase D and Phosphatidate, PA activity in a wide range of physiology, physiological processes and diseases, including inflammation, diabetes, phagocytosis, neurotransmission, neuronal & Electrical conduction system of the heart, cardiac signaling, and oncogenesis.


Function

Strictly speaking, phospholipase D is a transphosphatidylase: it mediates the exchange of polar headgroups covalently attached to membrane lipids, membrane-bound lipids. Utilizing water as a nucleophile, this enzyme catalyzes the bond cleavage, cleavage of the phosphodiester bond in structural phospholipids such as
phosphatidylcholine Phosphatidylcholines (PC) are a class of phospholipids that incorporate choline as a headgroup. They are a major component of biological membranes and can be easily obtained from a variety of readily available sources, such as egg yolk or soybeans, ...
and phosphatidylethanolamine. The products of this hydrolysis are the membrane-bound lipid phosphatidic acid (PA), and
choline Choline is an essential nutrient for humans and many other animals. Choline occurs as a cation An ion () is a particle, atom or molecule with a net electric charge, electrical charge. The charge of the electron is considered negative by ...
, which diffusion, diffuses into the cytosol. As
choline Choline is an essential nutrient for humans and many other animals. Choline occurs as a cation An ion () is a particle, atom or molecule with a net electric charge, electrical charge. The charge of the electron is considered negative by ...
has little second messenger activity, PLD activity is mostly signal transduction, transduced by the production of PA. PA is heavily involved in intracellular signal transduction. In addition, some members of the PLD superfamily (biology), superfamily may employ primary alcohols such as ethanol or 1-butanol in the cleavage of the phospholipid, effectively catalyzing the exchange the Chemical polarity, polar lipid, lipid headgroup. Other members of this family are able hydrolyze other phospholipid substrates, such as cardiolipin, or even the phosphodiester bond constituting the backbone of DNA.


Phosphatidic acid

Many of phospholipase D's Cell (biology)#Cellular processes, cellular functions are mediated by its principal product, phosphatidic acid (PA). Phosphatidic acid, PA is a electric charge, negatively charged phospholipid, whose small Phospholipid#Amphipathic character, head group promotes membrane curvature. It is thus thought to facilitate membrane-vesicle fusion and endocytosis, fission in a manner analogous to clathrin-mediated endocytosis. Phosphatidic acid, PA may also protein targeting, recruit proteins that contain its corresponding binding domain, a binding site, region characterized by basic (chemistry), basic amino acid-rich regions. Additionally, Phosphatidic acid, PA can be converted into a number of other lipids, such as lysophosphatidic acid (lyso-PA) or diacylglycerol, signal molecules which have a multitude of effects on Upstream and downstream (transduction), downstream metabolic pathway, cellular pathways.Phosphatidic acid, PA and its lipid derivatives are implicated in myriad biological process, processes that include intracellular vesicle trafficking, endocytosis, exocytosis, actin cytoskeleton, cytoskeleton dynamics, cell proliferation Cellular differentiation, differentiation, and cell migration, migration. Mammals, Mammalian PLD directly proteinā€“protein interaction, interacts with Protein kinase, kinases like Protein kinase C, PKC, Extracellular signal-regulated kinases, ERK, TYK and controls the signalling indicating that PLD is activated by these kinases. As
choline Choline is an essential nutrient for humans and many other animals. Choline occurs as a cation An ion () is a particle, atom or molecule with a net electric charge, electrical charge. The charge of the electron is considered negative by ...
is very abundant in the cell, PLD activity does not significantly affect choline levels, and choline is unlikely to play any role in signalling. Phosphatidic acid is a signal molecule and acts to recruit sphingosine kinase 1, SK1 to membranes. PA is extremely short lived and is rapidly hydrolysis, hydrolysed by the enzyme phosphatidate phosphatase to form diglyceride, diacylglycerol (DAG). DAG may also be converted to PA by diacylglycerol kinase, DAG kinase. Although PA and DAG are interconvertible, they do not act in the same metabolic pathway, pathways. Stimulus (physiology), Stimuli that Enzyme activator, activate PLD do not activate enzymes Upstream and downstream (transduction), downstream of DAG and vice versa. It is possible that, though PA and DAG are interconvertible, separate pools of signalling and non-signalling lipids may be maintained. Studies have suggested that DAG signalling is mediated by polyunsaturated DAG while PLD derived PA is monounsaturated or Saturated and unsaturated compounds, saturated. Thus functional saturated/monounsaturated PA can be degraded by hydrolysing it to form non-functional saturated/monounsaturated DAG while functional polyunsaturated DAG can be degraded by converting it into non-functional polyunsaturated PA. A lysophospholipase D called autotaxin was recently identified as having an important role in cell-proliferation through its product, lysophosphatidic acid (LPA).


Structure

Plant and animal PLDs have a consistent molecular structure, characterized by active site, sites of catalysis surrounded by an assortment of regulatory sequences. The active site of PLDs consists of four highly conserved sequence, conserved amino acid sequences (I-IV), of which Sequence motif, motifs II and IV are particularly conserved. These structural domains contain the distinguishing catalytic peptide sequence, sequence ''HxKxxxxD'' (HKD), where H, K, and D are the amino acids histidine (H), lysine (K), aspartic acid (D), while x represents nonconservative amino acids. These two HKD structural motif, motifs confer hydrolysis, hydrolytic activity to PLD, and are critical for its enzymatic activity both ''in vitro'' and ''in vivo''. Hydrolysis of the phosphodiester bond occurs when these HKD sequences are in the correct Enzyme catalysis#Proximity and orientation, proximity. Human proteins containing this motif include: * PGS1, PLD1, PLD2, PLD3, PLD4, PLD5 Phosphatidylcholine, PC-hydrolyzing PLD is a Protein superfamily#Structural homology, homologue of cardiolipin, cardiolipin synthase, phosphatidylserine synthase, bacterial PLDs, and viral proteins. Each of these appears to possess a Gene duplication, domain duplication which is apparent by the presence of two HKD structural motif, motifs containing well-conserved sequence, conserved histidine, lysine, and asparagine Residue (chemistry), residues which may contribute to the active site aspartic acid. An ''Escherichia coli'' endonuclease (nuc) and similar proteins appear to be PLD Protein superfamily#Structural homology, homologues but possess only one of these motifs. PLD genes additionally encode highly conserved allosteric regulation, regulatory regulatory sequence, domains: the PX domain, phox consensus sequence (PX), the pleckstrin homology domain (PH), and a binding site for phosphatidylinositol 4,5-bisphosphate (PIP2).


Mechanism of catalysis

PLD-Enzyme catalysis, catalyzed hydrolysis has been proposed to occur in two stages via a "ping-pong" mechanism. In this scheme, the histidine residues of each HKD motif successively nucleophilic addition, attack the phospholipid substrate. Functioning as nucleophiles, the constituent imidazole functional group, moieties of the histidines form transient covalent bonds with the phospholipid, producing a short-lived reaction intermediate, intermediate that can be easily hydrolysis, hydrolyzed by water in a subsequent Elementary reaction, step.


Mechanism of activation

Substrate presentation For mammalian PLD2, the molecular basis of activation is substrate presentation. The enzyme resides inactive in lipid micro-domains rich in sphingomyelin and depleted of PC substrate. Increased PIP2 or a decrease in cholesterol causes the enzyme to translocate to PIP2 micro domains near its substrate PC. Hence PLD can is primarily activated by localization within the plasma membrane rather than a protein conformational change. Disruption of lipid domains by anesthetics. or mechanical force. The protein may also undergo a conformational change upon PIP2 binding, but this has not been shown experimentally and would constitute a mechanism of activation distinct from substrate presentation.


Isoforms

Two major isoforms of phospholipase D has been identified in mammals, mammalian cells: PLD1 and PLD2 (53% Homology (biology)#Sequence homology, sequence homology), each encoded by distinct genes. PLD activity appears to be present in most cell types, with the possible exceptions of Peripheral blood lymphocyte, peripheral leukocytes and other lymphocytes. Both PLD isoforms require phosphatidylinositol 4,5-bisphosphate, PIP2 as a cofactor (biochemistry), cofactor for Enzyme catalysis, activity. PLD1 and PLD2 exhibit different subcellular localizations that dynamically change in the course of signal transduction. PLD activity has been observed within the plasma membrane, cytosol, Endoplasmic reticulum, ER, and Golgi complex.


PLD1

PLD1 is a 120 kDa protein that is mainly located on the Biological membrane, inner membranes of cells. It is primarily present at the Golgi complex, endosomes, lysosomes, and Granule (cell biology), secretory granules. Upon the Molecular binding, binding of an extracellular stimulus, PLD1 is Active transport, transported to the plasma membrane. Basal PLD1 activity is low however, and in order to signal transduction, transduce the extracellular signal, it must first be activation, activated by proteins such as ADP ribosylation factor, Arf, Rho family of GTPases, Rho, Rac (GTPase), Rac, and protein kinase C.


Regulation

The activity of phospholipase D is extensively allosteric regulation, regulated by hormones, neurotransmitters, lipids, Small GTPase, small monomeric GTPases, and other small molecules that molecular binding, bind to their corresponding binding domain, domains on the enzyme. In most cases, signal transduction is mediated through production of phosphatidic acid, which functions as a secondary messenger. Specific phospholipids are regulators of PLD activity in plant and animal cells. Most PLDs require phosphatidylinositol 4,5-bisphosphate (PIP2), as a cofactors for activity. phosphatidylinositol 4,5-bisphosphate, PIP2 and other phosphoinositides are important modifiers of
cytoskeletal Image:FluorescentCells.jpg, 300px, The eukaryotic cytoskeleton. Actin filaments are shown in red, and microtubules composed of beta tubulin are in green. The cytoskeleton is a complex, dynamic network of interlinking protein filaments present in t ...
dynamics and membrane transport and can traffic PLD to its substrate PC. PLDs regulated by these phospholipids are commonly involved in intracellular signal transduction. Their cooperative binding, activity is dependent upon the binding of these phosphoinositides near the active site. In plants and animals, this binding site is characterized by the presence of a conserved sequence of basic (chemistry), basic and aromaticity, aromatic amino acids. In plants such as ''Arabidopsis thaliana'', this peptide sequence, sequence is constituted by a ''RxxxxxKxR'' motif together with its inverted repeat, where R is arginine and K is lysine. Its Enzyme catalysis#Proximity and orientation, proximity to the active site ensures high level of PLD1 and PLD2 activity, and promotes the Protein translocation, translocation of PLD1 to protein targeting, target membranes in response to extracellular signals.


C2 domain

Calcium acts as a cofactor (biochemistry), cofactor in PLD isoforms that contain the C2 domain. Binding of Calcium in biology, Ca2+ to the C2 domain leads to conformational changes in the enzyme that strengthen Enzyme-substrate complex, enzyme-substrate binding, while weakening the Dissociation constant#Proteinā€“ligand binding, association with phosphoinositides. In some plant isoenzymes, such as PLDĪ², Calcium in biology, Ca2+ may bind directly to the active site, indirectly increasing its Chemical affinity, affinity for the substrate by strengthening the binding of the activator phosphatidylinositol 4,5-bisphosphate, PIP2.


PX domain

The PX domain, pbox consensus sequence (PX) is thought to mediate the binding of additional phosphatidylinositol phosphates, in particular, phosphatidylinositol 5-phosphate (PtdIns5P), a lipid thought to be required for endocytosis, may help facilitate the reinternalization of PLD1 from the plasma membrane.


PH domain

The highly conserved Pleckstrin homology domain (PH) is a structural domain approximately 120 amino acids in length. It binds phosphatidylinositol, phosphatidylinositides such as phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and phosphatidylinositol (4,5)-bisphosphate (PIP2). It may also bind heterotrimeric G proteins via their G beta-gamma complex, Ī²Ī³-subunit. Binding to this structural domain, domain is also thought to facilitate the endocytosis, re-internalization of the protein by increasing its Chemical affinity, affinity to endocytosis, endocytotic lipid rafts.


Interactions with small GTPases

In animal cells, small protein enzyme, factors are important additional Regulatory enzymes, regulators of PLD activity. These small GTPases, small monomeric GTPases are Protein family, members of the Rho family of GTPases, Rho and ADP ribosylation factor, ARF families of the Ras superfamily. Some of these proteins, such as Rac1, Cdc42, and RhoA, allosteric regulation, allosterically activate PLD1, mammalian PLD1, directly increasing its activity. In particular, the Protein translocation, translocation of cytosolic ADP-ribosylation factor (ARF) to the plasma membrane is essential for PLD activation.


Physiological and pathophysiological roles


Alcohol Intoxication

Phospholipase D metabolizes ethanol into phosphatidylethanol (PEtOH) in a process termed transphosphatidylation. Using fly genetics the PEtOH was shown to mediates alcohol's hyperactive response in fruit flies. And ethanol transphosphatidylation was shown to be up-regulated in alcoholics and the family members of alcoholic.s This ethanol transphosphatidylation mechanism recently emerged as an alternative theory for alcohol's effect on ion channels. Many ion channels are regulated by anionic lipids. and the competition of PEtOH with endogenous signaling lipids is thought to mediate the effect of ethanol on ion channels in some instances and not direct binding of the free ethanol to the channel.


In cancer

Phospholipase D is a regulator of several critical cellular processes, including vesicle transport, endocytosis, exocytosis, cell migration, and mitosis. Dysregulation of these biological processes, processes is commonplace in carcinogenesis, and in turn, Regulation of gene expression, abnormalities in PLD gene expression, expression have been implicated in the carcinogenesis, progression of several List of cancer types, types cancer. A Somatic evolution in cancer#Glossary, driver mutation conferring elevated PLD2 activity has been observed in several malignant breast cancers. Elevated PLD expression has also been correlated with Cancer staging, tumor size in colorectal carcinoma, gastric carcinoma, and renal cancer. However, the metabolic pathway, molecular pathways through which PLD drives cancer progression remain unclear. One potential hypothesis casts a critical role for phospholipase D in the activation of mTOR, a suppressor of cancer cell apoptosis. The ability of PLD to suppress apoptosis in cells with elevated tyrosine kinase activity makes it a candidate oncogene in cancers where such gene expression, expression is typical.


In neurodegenerative diseases

Phospholipase D may also play an important pathophysiology, pathophysiological role in the neurodegeneration, progression of Neurodegenerative disorders, neurodegenerative diseases, primarily through its capacity as a signal transduction, signal transducer in indispensable cellular processes like cytoskeleton, cytoskeletal reorganization and vesicle trafficking. Dysregulation of PLD by the protein alpha-synuclein, Ī±-synuclein has been shown to lead to the specific loss of Dopamine#In the brain, dopaminergic neurons in mammals. alpha-synuclein, Ī±-synuclein is the primary structural component of Lewy bodies, Protein aggregation, protein aggregates that are the hallmarks of Parkinson's disease. Disinhibition of PLD by alpha-synuclein, Ī±-synuclein may contribute to Parkinson's disease, Parkinson's deleterious phenotype. Abnormal PLD activity has also been suspected in Alzheimer's disease, where it has been observed to interact with presenilin 1 (PS-1), the principal component of the gamma secretase, Ī³-secretase Multiprotein complex, complex responsible for the proteolysis, enzymatic cleavage of amyloid precursor protein (APP). Extracellular Senile plaques, plaques of the product beta amyloid, Ī²-amyloid are a defining symptom, feature of Alzheimer's diseased brains. Action of PLD1 on PS-1 has been shown to affect the Protein targeting, intracellular trafficking of the amyloid precursor protein, amyloid precursor to this multiprotein complex, complex. Phospholipase D3 (PLD3), a non-classical and poorly characterized member of the PLD superfamily (biology), superfamily, has also been associated with the pathogenesis of this disease.


Gallery

Image:Phosphatidyl-Choline.svg, Phosphatidyl choline Image:Phosphatidate2.png, Phosphatidate Image:Choline-skeletal.svg, Choline Image:Phospholipases2.png, Phospholipase cleavage sites


References


External links

* {{Portal bar, Biology, border=no EC 3.1.4 Peripheral membrane proteins