Cytosolic Phospholipase A2 (pla2G4A)
The phospholipase A2 was the first of the phopsholipases to be identified when, in 1877, Bokay observed that the lecithin was degraded by a ferment obtained from pancreatic juice. Subsequently, in 1902, this enzyme, known as lecithinase, was detected also in cobra venom where it was observed to induce hemolytic activity through the lysis of erythrocytes membrane. The enzyme was then isolated from human pancreatic tissue by Gronchi and colleagues in 1936 (Glaser and Vadas 1995; Vance and Vance 2002). Since then, an increasing number of PLA2 have now been identified and grouped according to their biochemical features. However, the first evidence that mammalian cells contain a cytosolic calcium-dependent PLA2 able to specifically cleave arachidonic acid was reported only 50 years later by Flesch in 1985 (Flesch et al. 1985 ) and Alonso in 1986 (Alonso et al. 1986). Subsequently, important information regarding the role of cPLA2s in physiological processes and disease was provided by the knockout mouse model that revealed that cPLA2α knockout mice have normal growth and lifespan but exhibit an age-related renal dysfunction, ulcerative lesions of the small intestine, enlarged heart, and female reproduction defects demonstrating that metabolites generated by cPLA2s catalytic activity regulate several normal physiological processes (Bonventre et al. 1997; Downey et al. 2001; Uozumi et al. 1997; Takaku et al. 2000).
Phospholipases are a ubiquitous group of enzymes that hydrolyze phospholipids to generate molecules that may have potent biological activity. Phospholipids are dynamic molecules localized within lateral phases of membrane bilayers and in subcellular organelles. Phospholipids breakdown mediated by phospholipases generates both hydrophobic and hydrophilic molecules that can act at the site of production, at distal site within the cell or can be secreted and act outside the cell. Many of them exert their cellular function through extracellular or intracellular receptors. The classification of phospholipases is based on the site of attack. Phospholipases that catalyze the hydrolysis of the acyl-ester group are classified as phospholipase A (PLA): PLA1 hydrolizes the 1-acyl ester bond of phospholipids and PLA2 the 2-acyl ester bond. Some phospholipases hydrolize both acyl ester group and are known as phospholipase B. Phospholipase C (PLC) and phospholipase D (PLD) are both phosphodiesterases and catalyze the cleavage of glycerophosphate bond and the removal of the base group, respectively (Vance and Vance 2002). PLA2 that releases fatty acids from the second carbon group of glycerol specifically recognizes the sn-2 acyl bond of phospholipids and catalytically hydrolyzes the bond releasing arachidonic acid and lysophospholipids. In particular, arachidonic acid is converted in inflammatory mediators such as prostaglandins and leukotrienes which are categorized as potent inflammatory mediators implicated in many disorders such as asthma and arthritis. However, it can also directly modulate cellular function by altering membrane fluidity, activating protein kinases, and regulating gene transcription (Katsuki and Okuda 1995). On the other hand, lysophospholipids are involved in the control of phospholipid remodeling and membrane perturbation. Thus, PLA2 activity is tightly regulated in order to maintain levels of arachidonic acid and lysophospholipids necessary for the correct cellular homeostasis (Katsuki and Okuda 1995; Vance and Vance 2002). To date in humans 17 genes and 25 PLA2 isoforms have been identified. PLA2s can be distinct in groups on the base of their specific features such as sequence, molecular weight, disulfide bonding patters, and Ca2+ dependency. They are: (1) the secreted small molecular weight PLA2s (sPLA2s), (2) the Ca2+-independent PLA2s (iPLA2s), (3) the larger cytosolic Ca2+-dependent PLA2s (cPLA2s), (4) the platelet-activating factor-acetylhydrolases (PAF-AH), and (5) the lysosomal PLA2s. Differently from other PLA2 isoforms, considerable information is available about cPLA2 structure, function, and mechanisms of regulation (Gentile et al. 2012).
Nomenclature and Structure
Classical lipase activity is exerted by a catalytic domain containing a Ser-Asp/Gln-His triad; however, the group IV PLA2 family catalytic domain lacks of the His residue, thus it is composed by an unusual Ser-Asp dyad located in a deep cleft lined by hydrophobic residues. This funnel is covered by a flexible lid that moves to allow the access of the substrate to the active site (Dessen et al. 1999). This Ser-Asp dyad catalytic domain is highly conserved throughout evolution since it has been described also in the plant lipid acylhydrolase patatin and in phospholipase from Pseudomonas aeruginosa (Ghosh et al. 2006; Rydel et al. 2003). The serine (Ser228 in cPLA2α) is present in the pentapeptide sequence G-L-S-G-S that is similar to the lipase consensus sequence G-X-S-X-G. A conserved arginine (Arg200 in cPLA2α) is also required for catalysis (Dessen et al. 1999). Comparative structural analysis of the catalytic domain of the enzymes belonging to the group IV PLA2s revealed that it is essential for the arachidonoyl selectivity. In fact, the only differences into the catalytic domain are in two residues between cPLA2α and cPLA2γ. These differences may be responsible for the lack in specificity towards arachidonic acid and in sensitivity to inhibitors such as pyrrolidine-2 (Ghosh et al. 2006).
It is necessary the mobilization of intracellular calcium to obtain the maximal cathalytic activity of the enzyme. Calcium mobilization is mediated by an N-terminal calcium-dependent lipid-binding domain that colocalizes the catalytic domain with its membrane substrate (Ghosh et al. 2006). The calcium-binding domain of the cPLA2s is a classical C2 domain, present in a variety of mammals’ proteins, whose function is primarily to promote the interaction between protein and membrane. A C2 domain is composed of about 120 aminoacids that share a common fold of eight antiparallel β-sheet. Structural and functional analysis of the C2 domain of cPLA2α revealed that it contains three calcium-binding loops (CBL) that bind two calcium ions each through two acidic residues such as Asp and Asn. In the unbound state, the membrane-binding face of the C2 domain is electronegative due to the presence of these two residues, and do not interact with membranes. The binding with calcium ions determines the so-called electrostatic switch of the C2 domain that can bind anionic phospholipids in membranes. In particular, calcium binding to the C2 domain of cPLA2s promotes the interaction with phosphorylcholine rather than to anionic phospholipids. Alignment of the C2 domains of the members of the group IV of phospholipases revealed that cPLA2α contains seven calcium-binding residues, four of which are conserved in the other members of the group. The conserved residues are present in CBL1 and CBL 2 and are crucial for the binding to the membrane phospholipids. Another important structural difference between cPLA2α and the other members of the IV group is represented by the length of the linker that connects the catalytic domain to the C2 domain: in cPLA2α the two domains are connected by a 5-residues flexible linker that may undergo rotational changes affecting the interaction of the catalytic domain with the membrane, whereas in other cPLA2s the C2 and catalytic domain are connected by an approximately 120-residues linker that can influence both membrane-binding properties and enzyme tridimensional conformation (Dessen et al. 1999; Ghosh et al. 2006).
cPLA2α is encoded by a gene on human chromosome 1 next to the gene encoding COX2; it is ubiquitous and constitutively expressed in human cells. Its expression is enhanced by proinflammatory cytokines and growth factors and is inhibited by glucocorticoids as indicated by the presence of the INF-γ and glucocorticoid responsive elements on its promoter (Miyashita et al. 1995). Studies demonstrated that in smooth muscle cells cPLA2α expression is regulated by STAT-3 (Ghosh et al. 2006) and, moreover, that in several types of cancer it is overexpressed and upregulated by the oncogene ras through the phosphorylation of the kinases JNK and ERK (Van Putten et al. 2001).
cPLA2β gene is on human chromosome 15 near a gene cluster that encodes cPLA2γ, ε, and ζ and separated from this cluster by the genes Sptbn5 and Ehd4. These cPLA2s are highly homologous sharing 45–50% residues in the catalytic domain suggesting that they are arisen from an ancestral gene by duplication (Ohto et al. 2005). cPLA2β mRNA is widely expressed in human pancreas, liver, heart, and cerebellum, and its gene is immediately downstream of a complete JmjC domain which is a metalloenzyme present in nuclear protein with the ability to bind DNA. This implies that cPLA2β mRNA may undergo complex transcriptional and splicing regulation resulting in the production of diverse proteins. In fact, when the JmjC domain is completely transcribed, cPLA2β lacks C2 domain and is not able to bind membranes (Ghosh et al. 2006). cPLA2δ was found to be expressed in stratified squamous epithelium of the fetal skin and is significantly increased in the upper epidermis of psoriatic lesions. cPLA2δ gene encodes a 90 kDa protein which contains both C2 and catalytic domains (Chiba et al. 2004). cPLA2γ gene is on chromosome 19, and its mRNA is present in skeletal and cardiac muscle and in brain (Ghosh et al. 2006).
cPLA2s exert their activity mainly on membranes where they access the substrate. Upon stimulation, intracellular calcium concentration increases and induces the enzyme translocation from cytosol to membrane. cPLA2α has been shown to translocate primary to the perinuclear envelope, to Golgi, and to the endoplasmic reticulum. In particular, it has been shown that short-duration and low-concentration calcium transients induce translocation to Golgi, whereas high-concentration calcium transients induce translocation to ER. Moreover, CBL1 and CBL3 are critical for the specific targeting of cPLA2α to the Golgi apparatus. The localization to Golgi regulates Golgi architecture and membrane-trafficking events (Dessen et al. 1999). cPLA2α can localize also inside the nucleus as demonstrated by studies on endothelial cells. In particular, it has been shown that endothelial cells cycle entry is associated with release of high levels of arachidonic acid, which has been implicated in regulating cell proliferation (Herbert et al. 2005). Other sites of cPLA2α localization are represented by the membranes of the forming phagosomes in macrophages and by neutrophils membranes (Ghosh et al. 2006). Association of PLA2α with membranes is enhanced by several binding proteins such as vimentin, p11, annexin-1, caveolin-1, and cPLA2α-interacting protein PLIP (Ghosh et al. 2006). The localization of other members of the group IV PLA2s has been investigated by GFP-tagged proteins. This strategy demonstrated that cPLA2δ localizes to nuclear envelope in response to calcium ionophore and that cPLA2ε localizes to punctate structures in resting cells whereas neither cPLA2ε nor cPLA2ζ localize to membrane after calcium stimulation. Finally, cPLA2γ is constitutively bound to membrane of Golgi and ER since it contains a CAAX sequence in the C-terminus that is farnesylated (Ghosh et al. 2006). Moreover, studies in which cPLA2γ is overexpressed in COS cells revealed that it is bound to mitochondria suggesting that it can play a role in mitochondrial function such as initiating apoptosis (Duan et al. 2001).
cPLA2s activity is also regulated by phosphorylation. This molecular event is well studied for cPLA2α which contains several phosphorylation sites recognized by MAPKs, MNK-1, and CAMKII. Cellular studies demonstrated that phosphorylation at these sites only modestly increases arachidonic acid release after calcium transient increase, but induces a conformational change of the enzyme that enhances the interaction of the catalytic domain with its membrane substrate. However, phosphorylation is not sufficient to promote membrane binding in the absence of calcium increase (Ghosh et al. 2006).
Phospholipases are a ubiquitous group of enzymes that hydrolyze phospholipids to generate molecules that may have potent biological activity. Among phospholipases, phospholipase A2 releases fatty acids from the second carbon group of glycerol, specifically recognizes the sn-2-acyl bond of phospholipids, and catalytically hydrolyzes the bond releasing arachidonic acid and lysophospholipids. Arachidonic acid is then converted in inflammatory mediators such as prostaglandin and leukotrienes that are potent inflammatory mediators implicated in many disorders such as asthma and arthritis. PLA2s can be distinct in groups on the base of their specific features such as sequence, molecular weight, disulfide bonding patterns, and calcium dependency. The group of which considerable information are available about structure, function, and mechanisms of regulation are the large cytosolic calcium-dependent PLA2 (cPLA2). cPLA2s, also referred to as group IV PLA2, is a family of enzymes containing six members: cPLA2α, cPLA2β, cPLA2γ, cPLA2ε, and cPLA2ζ. X-ray crystal structure of cPLA2s revealed that they all contain an N-terminal calcium-dependent lipid binding/C2 domain that promotes interaction of the enzyme with membrane and a catalytic domain. The two domains are connected by a linker that has a variable length from 5 residues in the cPLA2α to 120 residues in the other members of the group. The C2 domains contain calcium-binding loops that bind calcium ions through the acidic residues Asp and Asn. The binding of these two residues with calcium ions determines the so-called electrostatic switch of the C2 domain that becomes electropositive and can bind anionic phospholipids in the membrane. The catalytic domain is represented by a Ser-Asp dyad located in a deep funnel lined by hydrophobic residues and covered by a flexible lid that moves to allow the access of the substrate to the active site. The regulation of cPLA2s activity is complex involving transcriptional and posttranscriptional processes, localization, phosphorylation, and intracellular calcium concentration increase. cPLA2s exert their activity mainly on membranes where they access the substrate. Upon stimulation, intracellular calcium concentration increases and induces the enzyme translocation from cytosol to membranes. These latter may be represented by nuclear envelope, Golgi and ER membranes, phagosomes, and plasma membranes. Recent studies revealed that cPLA2s are implicated also in regulating intracellular membrane trafficking being involved in the formation of carriers from donor membranes (Leslie 2015). cPLA2s activity is also regulated by phosphorylation even if phosphorylation alone is not sufficient to promote membrane binding in absence of calcium increase.
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