Calcium binding proteins and cellular regulation

An important feature of cellular regulation is the precise control of intracellular calcium levels. This is accomplished both by dynamic organelle release and sequestration of calcium and by specific calcium active transport mechanisms located in the plasma membrane. The actual calcium signal for mediation of a cellular response is carried out by specific intracellular proteins, the most widely studied examples are calmodulin and troponin C. The recent discovery of phospholipid protein kinase and calcimedins suggests receptor mediation via several independent proteins. The physiological importance of a particular protein as a calcium messenger rests on several features: 1) calcium binding is of the order of 1–10 μm, 2) the protein is known to be localized at the site of proposed action, 3) if translocation occurs upon activation, the time required is consistent with the time course of the physiologic response and 4) substrates or effectors at the next level of action when isolated can be demonstrated to have similar activation kinetics as $\text{in situ}$.     

Mammalian phospholipase D structure and regulation.

The recent identification of cDNA clones for phospholipase D1 and 2 has opened the door to new studies on its structure and regulation. PLD activity is encoded by at least two different genes that contain catalytic domains that relate their mechanism of action to phosphodiesterases. In vivo roles for PLD suggest that it may be important for multiple specialized steps in receptor dependent and constitutive processes of secretion, endocytosis, and membrane biogenesis.     

Potential anticoagulant activity of lipocortins and other calcium/phospholipid binding proteins

Thrombin generation was determined in the presence of phospholipids, coagulation factors Xm, Vm and II (prothrombin), calcium, and various calcium/phospholipid bindings proteins, including lipocortins I and II, 35 kDa calelectrin, and 32.5 kDa endonexin. All of these proteins induced a dose-dependent inhibition of thrombin generation similar to the inhibition of pig pancreas phospholipase A2. It is suggested that the ability of lipocortins and other related proteins to interact with anionic phospholipids in the presence of Ca++ is responsible for both their anticoagulant and anti-phospholipase A2 activity.     

The cellular retinoic acid binding proteins

The two cellular retinoic acid binding proteins, CRABP I and CRABP II, belong to a family of small cytosolic lipid binding proteins and are highly conserved during evolution. Both proteins are expressed during embryogenesis, particularly in the developing nervous system, craniofacial region and limb bud. CRABP I is also expressed in several adult tissues, however, in contrast, CRABP II expression appears to be limited to the skin. It is likely that these proteins serve as regulators in the transport and metabolism of retinoic acid in the developing embryo and throughout adult life. It has been proposed that CRABP I sequesters retinoic acid in the cytoplasm and prevents nuclear uptake of retinoic acid. A role in catabolism of retinoic acid has also been proposed. Recent gene targeting experiments have shown that neither of the two CRABPs are essential for normal embryonic development or adult life. Examination of CRABP I expression at subcellular resolution reveals a differential cytoplasmic and/or nuclear localization of the protein. A regulated nuclear uptake of CRABP I implies a role for this protein in the intracellular transport of retinoic acid. A protein mediated mechanism which controls the nuclear uptake of retinoic acid may play an important role in the transactivation of the nuclear retinoic acid receptors.     

Mechanisms of regulation of phospholipase D1 and D2 by the heterotrimeric G proteins G13 and Gq.

Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by alpha subunits of the heterotrimeric G proteins G(13) and G(q). Recently, we showed that rPLD1 is modified by Ser/Thr phosphorylation and palmitoylation. In this study, we first investigated the roles of these post-translational modifications on the activation of rPLD1 by constitutively active Galpha(13)Q226L and Galpha(q)Q209L. Mutations of Cys(240) and Cys(241) of rPLD1, which abolish both post-translational modifications, did not affect the ability of either Galpha(13)Q226L or Galpha(q)Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by Galpha(13)Q226L, although these mutant enzymes responded to phorbol ester and Galpha(q)Q209L. On the contrary, the PKC-insensitive mutant rPLD1(DeltaN168), which lacks the first 168 amino acids of rPLD1, responded to Galpha(13)Q226L but not to Galpha(q)Q209L. In addition, we found that rPLD2 was strongly activated by Galpha(q)Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by Galpha(13)Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of Galpha(q)Q209L or Galpha(13)Q226L. The suppressive effect of Galpha(13)Q226L on rPLD2 was reversed by dominant negative N19RhoA and the C3 exoenzyme of Clostridium botulinum, further supporting a role for RhoA. In summary, Galpha(13) activation of rPLD1 in COS-7 cells is mediated by Rho, while Galpha(q) activation requires PKC. rPLD2 is activated by Galpha(q), but is inhibited by Galpha(13). Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.     

N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: a novel enzyme of the beta-lactamase fold family releasing anandamide and other N-acylethanolamines

N-acylethanolamines (NAEs) are a lipid class present in brain and other animal tissues and contains anandamide (an endocannabinoid) and other bioactive substances. NAEs are formed from N-acylphosphatidylethanolamines (NAPEs) by a phospholipase D (PLD)-type enzyme abbreviated to NAPE-PLD. Although this enzyme has been recognized for more than 20 years, its molecular cloning has only recently been achieved by us. We highly purified NAPE-PLD from the particulate fraction of rat heart, and on the basis of peptide sequences with the purified enzyme cloned its cDNA from mouse, rat and human. The deduced primary structures revealed no homology with any PLDs so far reported, but was suggested to belong to the beta-lactamase fold family. When overexpressed in COS-7 cells, the NAPE-PLD activity increased about 1000-fold in comparison with the endogenous activity. The recombinant enzyme generated various long-chain NAEs including anandamide from their corresponding NAPEs at similar rates. However, the enzyme was inactive with phosphatidylethanolamine and phosphatidylcholine and did not catalyze transphosphatidylation, a reaction characteristic of PLD. The enzyme was widely expressed in murine organs with higher levels in brain, testis and kidney. The existence of NAPE-PLD specifically hydrolyzing NAPEs to NAEs emphasizes physiological significance of NAEs including anandamide in brain and other tissues.     

Activation of phospholipase D by PKC and GTPgammaS in human neuroblastoma cells overexpressing MARCKS

Regulation of phospholipase D (PLD) activity participating in signal transduction involves complex interactions with small G-proteins (ARF, Rho) and protein kinase C isoforms (PKCalpha). In SK-N-MC human neuroblastoma cells, phorbol ester (TPA) activation of PLD was enhanced by overexpressing myristoylated alanine-rich C kinase substrate (MARCKS). To study MARCKS interactions with PLD, we investigated PLD isoform expression and activation by TPA and GTPgammaS in intact and digitonin-permeabilized clones transfected with MARCKS (M22). PLD2 was in both cytosol and membrane fractions while PLD1 was primarily membrane-associated in both vector control and M22 cells; location or quantities were unaltered by TPA treatment. TPA-stimulated PLD activity was higher in both intact and digitonin-permeabilized M22 cells than in vector controls. In contrast, GTPgammaS-stimulated PLD activity was independent of MARCKS expression but was additive with MARCKS-PKC-dependent activation in permeabilized cells. Combinations of PKC inhibition and down-regulation in intact and permeabilized (with GTPgammaS present) cells indicated that a PKC-mediated phosphorylation event was necessary in intact cells without access to GTPgammaS, stimulation of PLD mediated by GTPgammaS was independent of PKC, and PLD activation by PKC in permeabilized cells was kinase-independent. Western blot analysis showed that MARCKS, PKCalpha, PLD1 and PLD2 were present in a detergent-insoluble fraction (DIF); GTPgammaS increased recovery of PLD2 in DIF. Disruption of cholesterol-rich DIFs with digitonin, cyclodextrin or filipin potentiated activation of PLD by TPA. Our studies suggest that activation of PLD by PKC requires MARCKS and can involve both phosphorylation-independent and -dependent processes. As PLD activation by GTPgammaS is PKC-MARCKS-independent, MARCKS may provide a fine tuning component in conjunction with G-protein-mediated mechanisms for regulation of PLD.     

D5 dopamine receptor regulation of phospholipase D

D(1)-like receptors have been reported to decrease oxidative stress in vascular smooth muscle cells by decreasing phospholipase D (PLD) activity. However, the PLD isoform regulated by D(1)-like receptors (D(1) or D(5)) and whether abnormal regulation of PLD by D(1)-like receptors plays a role in the pathogenesis of hypertension are unknown. The hypothesis that the D(5) receptor is the D(1)-like receptor that inhibits PLD activity and serves to regulate blood pressure was tested using D(5) receptor mutant mice (D(5)(-/-)). We found that in the mouse kidney, PLD2, like the D(5) receptor, is mainly expressed in renal brush-border membranes, whereas PLD1 is mainly expressed in renal vessels with faint staining in brush-border membranes and collecting ducts. Total renal PLD activity is increased in D(5)(-/-) mice relative to congenic D(5) wild-type (D(5)(+/+)) mice. PLD2, but not PLD1, expression is greater in D(5)(-/-) than in D(5)(+/+) mice. The D(5) receptor agonist fenoldopam decreases PLD2, but not PLD1, expression and activity in human embryonic kidney-293 cells heterologously expressing the human D(5) receptor, effects that are blocked by the D(5) receptor antagonist SCH-23390. These studies show that the D(5) receptor regulates PLD2 activity and expression. The hypertension in the D(5)(-/-) mice is associated with increased PLD expression and activity. Impaired D(5) receptor regulation of PLD2 may play a role in the pathogenesis of hypertension.     

A possible structural model of members of the CPF family of cuticular proteins implicating binding to components other than chitin

The physical properties of cuticle are determined by the structure of its two major components, cuticular proteins (CPs) and chitin, and, also, by their interactions.A common consensus region (extended R&R Consensus) found in the majority of cuticular proteins, the CPRs, binds to chitin. Previous work established that β-pleated sheet predominates in the Consensus region and we proposed that it is responsible for the formation of helicoidal cuticle. Remote sequence similarity between CPRs and a lipocalin, bovine plasma retinol binding protein (RBP), led us to suggest an antiparallel β-sheet half-barrel structure as the basic folding motif of the R&R Consensus. There are several other families of cuticular proteins. One of the best defined is CPF. Its four members in Anopheles gambiae are expressed during the early stages of either pharate pupal or pharate adult development, suggesting that the proteins contribute to the outer regions of the cuticle, the epi- and/or exo-cuticle. These proteins did not bind to chitin in the same assay used successfully for CPRs. Although CPFs are distinct in sequence from CPRs, the same lipocalin could also be used to derive homology models for one A. gambiae and one Drosophila melanogaster CPF. For the CPFs, the basic folding motif predicted is an eight-stranded, antiparallel β-sheet, full-barrel structure. Possible implications of this structure are discussed and docking experiments were carried out with one possible Drosophila ligand, 7(Z),11(Z)-heptacosadiene.     

The regulation of phospholipase D by inositol phospholipids and small GTPases

Phospholipase D1 and D2 (PLD1, PLD2) both have PX and PH domains in their N-terminal regions with these inositol lipid binding domains playing key roles in regulating PLD activity and localisation. The activity of PLD1 is also regulated by protein kinase C and members of the Rho and Arf families of GTPases. Each of these proteins binds to unique sites; however, there appears to be little in vitro discrimination between individual family members. In agonist-stimulated cells, however, there is specificity, with, for example in RBL-2H3 cells, antigen stimulating the activation of PLD1 by association with Arf6, Rac1 and protein kinase Calpha. PLD2 appears to be less directly regulated by GTPases and rather is primarily controlled through interaction with phosphatidylinositol 4-phosphate 5-kinase that generates the activating phosphatidylinositol 4,5-bisphosphate.     

Differential mechanisms of retinoid transfer from cellular retinol binding proteins types I and II to phospholipid membranes

Cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II) are known to differentially facilitate retinoid metabolism by several membrane-associated enzymes. The mechanism of ligand transfer to phospholipid small unilamellar vesicles was compared in order to determine whether differences in ligand trafficking properties could underlie these functional differences. Unidirectional transfer of retinol from the CRBPs to membranes was monitored by following the increase in intrinsic protein fluorescence that occurs upon ligand dissociation. The results showed that ligand transfer of retinol from CRBP-I was >5-fold faster than transfer from CRBP-II. For both proteins, transfer of the other naturally occurring retinoid, retinaldehyde, was 4-5-fold faster than transfer of retinol. Rates of ligand transfer from CRBP-I to small unilamellar vesicles increased with increasing concentration of acceptor membrane and with the incorporation of the anionic lipids cardiolipin or phosphatidylserine into membranes. In contrast, transfer from CRBP-II was unaffected by either membrane concentration or composition. Preincubation of anionic vesicles with CRBP-I was able to prevent cytochrome c, a peripheral membrane protein, from binding, whereas CRBP-II was ineffective. In addition, monolayer exclusion experiments demonstrated differences in the rate and magnitude of the CRBP interactions with phospholipid membranes. These results suggest that the mechanisms of ligand transfer from CRBP-I and CRBP-II to membranes are markedly different as follows: transfer from CRBP-I may involve and require effective collisional interactions with membranes, whereas a diffusional process primarily mediates transfer from CRBP-II. These differences may help account for their distinct functional roles in the modulation of intracellular retinoid metabolism.     

Multiple forms of phospholipase D in plants: the gene family, catalytic and regulatory properties, and cellular functions

Multiple Phospholipase D (PLD) genes have been identified in plants and encode isoforms with distinct regulatory and catalytic properties. Elucidation of the genetic and biochemical heterogeneity has provided important clues as to the regulation and function of this family of enzymes. Polyphosphoinositides, Ca(2+), and G-proteins are possible cellular regulators for PLD activation. PLD-mediated hydrolysis of membrane lipids increases in response to various stresses. Recent studies suggest that PLD plays a role in the signaling and production of hormones involved in plant stress responses.     

Functional anatomy of phospholipid binding and regulation of phosphoinositide homeostasis by proteins of the sec14 superfamily

Sec14, the major yeast phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein, regulates essential interfaces between lipid metabolism and membrane trafficking from the trans-Golgi network (TGN). How Sec14 does so remains unclear. We report that Sec14 binds PtdIns and PtdCho at distinct (but overlapping) sites, and both PtdIns- and PtdCho-binding activities are essential Sec14 activities. We further show both activities must reside within the same molecule to reconstitute a functional Sec14 and for effective Sec14-mediated regulation of phosphoinositide homeostasis in vivo. This regulation is uncoupled from PtdIns-transfer activity and argues for an interfacial presentation mode for Sec14-mediated potentiation of PtdIns kinases. Such a regulatory role for Sec14 is a primary counter to action of the Kes1 sterol-binding protein that antagonizes PtdIns 4-OH kinase activity in vivo. Collectively, these findings outline functional mechanisms for the Sec14 superfamily and reveal additional layers of complexity for regulating phosphoinositide homeostasis in eukaryotes.     

Dodecins: a family of lumichrome binding proteins

Dodecin is a small dodecameric flavoprotein from Halobacterium salinarum that contains two flavins stacked between two tryptophan residues to form an aromatic tetrade. The functional properties of heterologously expressed dodecin were investigated by fluorescence spectroscopy, which allowed the determination of dissociation constants for a number of protein-ligand complexes. The values obtained were in the nanomolar to micromolar range and correlate positively with the ligand size. These data were supplemented by X-ray crystal structures of the apododecin and holocomplexes with lumichrome, lumiflavin, riboflavin and FMN at resolutions between 1.55 to 1.95 A to unravel a gating mechanism as the structural basis for the preferential binding of the small ligands lumichrome and lumiflavin. The detailed analysis of the dodecin manifold for preferential binding of lumichrome and lumiflavin provides insight on a subatom level into a protein's strategy to gain selectivity for low molecular mass compounds by steric restrictions rather than specific interactions. Investigations on the ligand composition of a wild-type dodecin crystal (1.32 A resolution) support conclusions of functional and structural investigations on heterologously expressed dodecin, and strongly suggest that lumichrome, a molecule associated with the flavin metabolism, is a ligand of dodecin in vivo. Studies on mutant protein and a Halorhodospira halophila homologue spread the idea of a lumichrome binding system as a possible "waste"-trapping device, widely distributed in prokaryotes.