| Phospholipase D | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| Symbol | PLDc | ||||||||
| Pfam | PF03009 | ||||||||
| InterPro | IPR001736 | ||||||||
| SMART | SM00155 | ||||||||
| PROSITE | PDOC50035 | ||||||||
| SCOP2 | 1byr /SCOPe /SUPFAM | ||||||||
| OPM superfamily | 118 | ||||||||
| OPM protein | 3rlh | ||||||||
| CDD | cd00138 | ||||||||
| Membranome | 306 | ||||||||
| |||||||||
| phospholipase D | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| EC no. | 3.1.4.4 | ||||||||
| CAS no. | 9001-87-0 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDBPDBePDBsum | ||||||||
| Gene Ontology | AmiGO /QuickGO | ||||||||
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Phospholipase D (EC 3.1.4.4,lipophosphodiesterase II, lecithinase D, choline phosphatase, PLD; systematic namephosphatidylcholine phosphatidohydrolase) is an anesthetic sensitive[1] and mechanosensitive[2]enzyme of thephospholipasesuperfamily that catalyses the following reaction
Phospholipases occur widely, and can be found in a wide range of organisms, including bacteria, yeast, plants, animals, and viruses.[3][4] Phospholipase D's principalsubstrate isphosphatidylcholine, which ithydrolyzes to produce thesignal moleculephosphatidic acid (PA), andsolublecholine in a cholesterol dependent process calledsubstrate presentation.[5] Plants contain numerous genes that encode various PLDisoenzymes, withmolecular weights ranging from 90 to 125kDa.[6] Mammalian cells encode two isoforms of phospholipase D:PLD1 andPLD2.[7] Phospholipase D is an important player in manyphysiological processes, includingmembrane trafficking,cytoskeletal reorganization,receptor-mediated endocytosis,exocytosis, andcell migration.[8] Through these processes, it has been further implicated in thepathophysiology of multiplediseases: in particular the progression ofParkinson's andAlzheimer's, as well as variouscancers.[6][8] PLD may also help set the threshold for sensitivity to anesthesia and mechanical force.[9][10]
PLD-typeactivity was first reported in 1947 by Donald J. Hanahan and I.L. Chaikoff.[3] It was not until 1975, however, that thehydrolytic mechanism of action was elucidated inmammalian cells. Plantisoforms of PLD were firstpurified from cabbage andcastor bean;PLDα was ultimatelycloned and characterized from a variety of plants, including rice, corn, and tomato.[3] Plant PLDs have been cloned in three isoforms:PLDα,PLDβ, andPLDγ.[11]More than half a century of biochemical studies have implicated phospholipase D andPA activity in a wide range ofphysiological processes anddiseases, includinginflammation,diabetes,phagocytosis,neuronal &cardiac signaling, andoncogenesis.[12]
Strictly speaking, phospholipase D is atransphosphatidylase: it mediates the exchange of polar headgroups covalently attached tomembrane-bound lipids. Utilizing water as anucleophile, this enzyme catalyzes thecleavage of thephosphodiester bond in structuralphospholipids such asphosphatidylcholine andphosphatidylethanolamine.[6] The products of thishydrolysis are the membrane-boundlipidphosphatidic acid (PA), andcholine, whichdiffuses into thecytosol. Ascholine has littlesecond messenger activity, PLD activity is mostlytransduced by the production of PA.[8][13] PA is heavily involved inintracellularsignal transduction.[14] In addition, some members of the PLDsuperfamily may employprimary alcohols such asethanol or1-butanol in the cleavage of thephospholipid, effectively catalyzing the exchange thepolarlipid headgroup.[6][11] Other members of this family are ablehydrolyze other phospholipid substrates, such ascardiolipin, or even thephosphodiester bond constituting the backbone ofDNA.[7]
Many of phospholipase D'scellular functions are mediated by its principal product,phosphatidic acid (PA).PA is anegatively chargedphospholipid, whose smallhead group promotesmembrane curvature.[7] It is thus thought to facilitatemembrane-vesicle fusion andfission in a manner analogous toclathrin-mediated endocytosis.[7]PA may alsorecruit proteins that contain its correspondingbinding domain, aregion characterized bybasicamino acid-rich regions. Additionally,PA can be converted into a number of otherlipids, such aslysophosphatidic acid (lyso-PA) ordiacylglycerol,signal molecules which have a multitude of effects ondownstreamcellular pathways.[11]PA and itslipid derivatives are implicated in myriadprocesses that includeintracellularvesicle trafficking,endocytosis,exocytosis,actincytoskeleton dynamics,cell proliferationdifferentiation, andmigration.[7]
Mammalian PLD directlyinteracts withkinases likePKC,ERK,TYK and controls the signalling indicating that PLD is activated by these kinases.[15] Ascholine 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 asignal molecule and acts to recruitSK1 tomembranes. PA is extremely short lived and is rapidlyhydrolysed by the enzymephosphatidate phosphatase to formdiacylglycerol (DAG). DAG may also be converted to PA byDAG kinase. Although PA and DAG are interconvertible, they do not act in the samepathways.Stimuli thatactivate PLD do not activate enzymesdownstream of DAG and vice versa.
It is possible that, though PA and DAG are interconvertible, separate pools of signalling and non-signallinglipids may be maintained. Studies have suggested that DAG signalling is mediated bypolyunsaturated DAG while PLD derived PA ismonounsaturated orsaturated. 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.[16][17][18]
A lysophospholipase D calledautotaxin was recently identified as having an important role in cell-proliferation through its product,lysophosphatidic acid (LPA).
Plant and animal PLDs have a consistentmolecular structure, characterized bysites of catalysis surrounded by an assortment ofregulatory sequences.[6] Theactive site of PLDs consists of four highlyconservedamino acidsequences (I-IV), of whichmotifs II and IV are particularly conserved. Thesestructural domains contain the distinguishing catalyticsequenceHxKxxxxD (HKD), whereH,K, andD are the amino acidshistidine (H),lysine (K),aspartic acid (D), while x represents nonconservativeamino acids.[6][7] These two HKDmotifs conferhydrolytic activity to PLD, and are critical for its enzymatic activity bothin vitro andin vivo.[7][12]Hydrolysis of thephosphodiester bond occurs when these HKD sequences are in the correctproximity.
Human proteins containing this motif include:
PC-hydrolyzing PLD is ahomologue ofcardiolipin synthase,[19][20]phosphatidylserine synthase,bacterial PLDs, andviral proteins. Each of these appears to possess adomain duplication which is apparent by the presence of two HKDmotifs containing well-conservedhistidine,lysine, andasparagineresidues which may contribute to theactive siteaspartic acid. AnEscherichia coliendonuclease (nuc) and similar proteins appear to be PLDhomologues but possess only one of these motifs.[21][22][23][24]
PLDgenes additionally encode highly conservedregulatorydomains: thephox consensus sequence (PX), thepleckstrin homology domain (PH), and a binding site forphosphatidylinositol 4,5-bisphosphate (PIP2).[4]
PLD-catalyzedhydrolysis has been proposed to occur in two stages via a "ping-pong" mechanism. In this scheme, thehistidine residues of each HKD motif successivelyattack thephospholipidsubstrate. Functioning asnucleophiles, the constituentimidazolemoieties of thehistidines form transientcovalent bonds with thephospholipid, producing a short-livedintermediate that can be easilyhydrolyzed by water in a subsequentstep.[6][14]
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.[25] 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.[26] or mechanical force.[25] 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.
Two majorisoforms of phospholipase D has been identified inmammalian cells:PLD1 andPLD2 (53%sequence homology),[27] each encoded by distinctgenes.[7] PLD activity appears to be present in mostcell types, with the possible exceptions ofperipheral leukocytes and otherlymphocytes.[12] Both PLD isoforms requirePIP2 as acofactor foractivity.[7]PLD1 andPLD2 exhibit differentsubcellular localizations that dynamically change in the course ofsignal transduction. PLD activity has been observed within theplasma membrane,cytosol,ER, andGolgi complex.[12]
PLD1 is a 120 kDa protein that is mainly located on theinner membranes of cells. It is primarily present at theGolgi complex,endosomes,lysosomes, andsecretory granules.[7] Upon thebinding of anextracellular stimulus,PLD1 istransported to theplasma membrane. Basal PLD1 activity is low however, and in order totransduce the extracellular signal, it must first beactivated byproteins such asArf,Rho,Rac, andprotein kinase C.[7][8][13]
PLD2[edit]Main article:PLD2 In contrast, PLD2 is a 106kDa protein that primarilylocalizes to theplasma membrane, residing in light membranelipid rafts.[6][8] It has high intrinsic catalytic activity, and is only weakly activated by the above molecules.[6] |
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The activity of phospholipase D is extensivelyregulated byhormones,neurotransmitters,lipids,small monomeric GTPases, and other small molecules thatbind to their correspondingdomains on the enzyme.[6] In most cases,signal transduction is mediated through production ofphosphatidic acid, which functions as asecondary messenger.[6]
Specificphospholipids are regulators of PLD activity in plant and animal cells.[3][6] Most PLDs requirephosphatidylinositol 4,5-bisphosphate (PIP2), as a cofactors for activity.[4][6]PIP2 and otherphosphoinositides are important modifiers ofcytoskeletal dynamics andmembrane transport and can traffic PLD to its substrate PC.[28] PLDs regulated by thesephospholipids are commonly involved inintracellularsignal transduction.[6] Theiractivity is dependent upon the binding of thesephosphoinositides near theactive site.[6] In plants and animals, this binding site is characterized by the presence of aconserved sequence ofbasic andaromaticamino acids.[6][14] In plants such asArabidopsis thaliana, thissequence is constituted by aRxxxxxKxR motif together with itsinverted repeat, whereR isarginine andK islysine. Itsproximity to theactive site ensures high level ofPLD1 andPLD2 activity, and promotes thetranslocation of PLD1 totarget membranes in response toextracellular signals.[6]
Calcium acts as acofactor in PLDisoforms that contain theC2 domain. Binding ofCa2+ to theC2 domain leads toconformational changes in the enzyme that strengthenenzyme-substrate binding, while weakening theassociation withphosphoinositides. In some plantisoenzymes, such asPLDβ,Ca2+ may bind directly to theactive site, indirectly increasing itsaffinity for thesubstrate by strengthening the binding of the activatorPIP2.[6]
Thepbox 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 forendocytosis, may help facilitate the reinternalization ofPLD1 from theplasma membrane.[3]
The highly conservedPleckstrin homology domain (PH) is astructural domain approximately 120amino acids in length. It bindsphosphatidylinositides such asphosphatidylinositol (3,4,5)-trisphosphate (PIP3) andphosphatidylinositol (4,5)-bisphosphate (PIP2). It may also bindheterotrimeric G proteins via theirβγ-subunit. Binding to thisdomain is also thought to facilitate there-internalization of the protein by increasing itsaffinity toendocytoticlipid rafts.[3]
Inanimal cells, small proteinfactors are important additionalregulators of PLD activity. Thesesmall monomeric GTPases aremembers of theRho andARF families of theRas superfamily. Some of these proteins, such asRac1,Cdc42, andRhoA,allosterically activatemammalian PLD1, directly increasing its activity. In particular, thetranslocation ofcytosolicADP-ribosylation factor (ARF) to theplasma membrane is essential for PLD activation.[3][6]
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.[29] And ethanol transphosphatidylation was shown to be up-regulated in alcoholics and the family members of alcoholic.s[30] 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.[31] 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.[29]
PLD2 is a mechanosensor and directly sensitive to mechanical disruption of clustered GM1 lipids.[5] Mechanical disruption (fluid shear) then signals for the cell to differentiate. PLD2 also activates TREK-1 channels, a potassium channel in the analgesic pathway.[32]
PLD2 is upstream of Piezo2 and inhibits the channel.[33] Piezo2 is an excitatory channel, ence PLD inhibits an excitatory channel and activates TREK-1 which is an inhibitory channel. The channels combine to reduce neuronal excitability.
Phospholipase D is a regulator of several critical cellular processes, includingvesicle transport,endocytosis,exocytosis,cell migration, andmitosis.[8]Dysregulation of theseprocesses is commonplace incarcinogenesis,[8] and in turn,abnormalities in PLDexpression have been implicated in theprogression of severaltypescancer.[4][7] Adriver mutation conferring elevated PLD2 activity has been observed in severalmalignantbreast cancers.[7] Elevated PLD expression has also been correlated withtumor size incolorectal carcinoma,gastric carcinoma, andrenal cancer.[7][8] However, themolecular pathways through which PLD drives cancer progression remain unclear.[7] One potentialhypothesis casts a critical role for phospholipase D in the activation ofmTOR, a suppressor ofcancer cellapoptosis.[7] The ability of PLD to suppressapoptosis in cells with elevatedtyrosine kinase activity makes it a candidateoncogene incancers where suchexpression is typical.[8]
Phospholipase D may also play an importantpathophysiological role in theprogression ofneurodegenerative diseases, primarily through its capacity as asignal transducer in indispensablecellular processes likecytoskeletal reorganization andvesicle trafficking.[27]Dysregulation of PLD by the proteinα-synuclein has been shown to lead to the specific loss ofdopaminergicneurons inmammals.α-synuclein is the primary structural component ofLewy bodies,protein aggregates that are the hallmarks ofParkinson's disease.[7] Disinhibition of PLD byα-synuclein may contribute toParkinson's deleteriousphenotype.[7]
Abnormal PLD activity has also been suspected inAlzheimer's disease, where it has been observed to interact withpresenilin 1 (PS-1), the principal component of theγ-secretasecomplex responsible for theenzymatic cleavage ofamyloid precursor protein (APP).Extracellularplaques of the productβ-amyloid are a definingfeature ofAlzheimer's diseased brains.[7] Action ofPLD1 on PS-1 has been shown to affect theintracellular trafficking of theamyloid precursor to thiscomplex.[7][27] Phospholipase D3 (PLD3), a non-classical and poorly characterized member of the PLDsuperfamily, has also been associated with thepathogenesis of this disease.[34]
