VCAM-1 as a predictor biomarker in cardiovascular disease

The vascular cellular adhesion molecule-1 (VCAM-1) is a protein that canonically participates in the adhesion and transmigration of leukocytes to the interstitium during inflammation. VCAM-1 expression, together with soluble VCAM-1 (sVCAM-1) induced by the shedding of VCAM-1 by metalloproteinases, have been proposed as biomarkers in immunological diseases, cancer, autoimmune myocarditis, and as predictors of mortality and morbidity in patients with chronic heart failure (HF), endothelial injury in patients with coronary artery disease, and arrhythmias. This revision aims to discuss the role of sVCAM-1 as a biomarker to predict the occurrence, development, and preservation of cardiovascular disease.     

Phosphatidylinositol 4,5-Bisphosphate Decreases the Concentration of Ca2+, Phosphatidylserine and Diacylglycerol Required for Protein Kinase C α to Reach Maximum Activity

The C2 domain of PKCα possesses two different binding sites, one for Ca²⁺ and phosphatidylserine and a second one that binds PIP₂ with very high affinity. The enzymatic activity of PKCα was studied by activating it with large unilamellar lipid vesicles, varying the concentration of Ca²⁺ and the contents of dioleylglycerol (DOG), phosphatidylinositol 4,5-bisphosphate (PIP₂) and phosphadidylserine (POPS) in these model membranes. The results showed that PIP₂ increased the V_max of PKCα and, when the PIP₂ concentration was 5 mol% of the total lipid in the membrane, the addition of 2 mol% of DOG did not increase the activity. In addition PIP₂ decreases K_0.5 of Ca²⁺ more than 3-fold, that of DOG almost 5-fold and that of POPS by a half. The K_0.5 values of PIP₂ amounted to only 0.11 µM in the presence of DOG and 0.39 in its absence, which is within the expected physiological range for the inner monolayer of a mammalian plasma membrane. As a consequence, PKCα may be expected to operate near its maximum capacity even in the absence of a cell signal producing diacylglycerol. Nevertheless, we have shown that the presence of DOG may also help, since the K_0.5 for PIP₂ notably decreases in its presence. Taken together, these results underline the great importance of PIP₂ in the activation of PKCα and demonstrate that in its presence, the most important cell signal for triggering the activity of this enzyme is the increase in the concentration of cytoplasmic Ca²⁺.     

Identification of the phosphatidylserine binding site in the C2 domain that is important for PKC alpha activation and in vivo cell localization

The C2 domain of classical PKCs binds to membranes through Ca(2+) bridging to phosphatidylserine as recently observed through X-ray diffraction of the isolated domain. Additionally, it has been proposed that N189, T251, R216, and R249A interact directly with phosphatidylserine [Verdaguer, N., et al. (1999) EMBO J. 18, 6329-6338]. When these four residues were mutated to Ala to determine their role in PKC binding to phospholipid membranes, PKC activation, and in its in vivo localization, the results revealed that they were very important for the activation of full-length PKCalpha. N189, in particular, was involved in the activation of the enzyme after its interaction with PS, since its mutation to Ala did not decrease the level of membrane binding but did prevent full enzyme activation. On the other hand, mutations R216A, R249A, and T251A affected both membrane binding and enzyme activation, although T251A had the most drastic effect, suggesting that the protein interactions with the carbonyl groups of the phospholipid are also a key event in the activation process. Taken together, these results show that the four residues located near the calcium binding site are critical in phosphatidylserine-dependent PKCalpha activation, in which N189 plays an important role, triggering the enzyme activation probably by interacting with neighboring residues of the protein when lipid binding occurs. Furthermore, these results provide strong evidence for better defining one of the two phosphatidylserine isomer models proposed in the previous crystallographic report.     

Retinoic acid binds to the C2-domain of protein kinase C(alpha)

Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity.     

Chronic hyperammonemia alters protein phosphorylation and glutamate receptor-associated signal transduction in brain

There is substantial evidence that hyperammonemia is one of the main factors contributing to the neurological alterations found in hepatic encephalopathy. The mechanisms by which chronic moderate hyperammonemia affects brain function involves alterations in neurotransmission at different steps. This article reviews the effects of hyperammonemia on phosphorylation of key brain proteins involved in neurotransmission (the microtubule-associated protein (MAP-2), Na+/K+-ATPase and NMDA receptors). The physiological function of these proteins is modulated by phosphorylation and its altered phosphorylation in hyperammonemia may contribute to impairment of neurotransmission. The effects of chronic hyperammonemia on signal transduction pathways associated to glutamate receptors, such as the glutamate-nitric oxide (NO)-cGMP pathway, are also reviewed. The possible contribution of the impairment of this pathway in brain in vivo to the neurological alterations present in patients with hepatic encephalopathy is discussed.     

The C2 domain of protein kinase calpha is directly involved in the diacylglycerol-dependent binding of the C1 domain to the membrane

Protein kinase Calpha (PKCalpha), which is known to be critical for the control of many cellular processes, was submitted to site-directed mutagenesis in order to test the functionality of several amino acidic residues. Thus, D187, D246 and D248, all of which are located at the Ca(2+) binding site of the C2 domain, were substituted by N. Subcellular fractionation experiments demonstrated that these mutations are important for both Ca(2+)-dependent and diacylglycerol-dependent membrane binding. The mutants are not able to phosphorylate typical PKC substrates, such as histone and myelin basic protein. Furthermore, using increasing concentrations of dioleylglycerol, one of the mutants (D246/248N) was able to recover total activity although the amounts of dioleylglycerol it required were larger than those required by wild type protein. On the other hand, the other mutants (D187N and D187/246/248) only recovered 50% of their activity. These data suggest that there is a relationship between the C1 domain, where dioleylglycerol binds, and the C2 domain, and that this relationship is very important for enzyme activation. These findings led us to propose a mechanism for PKCalpha activation, where C1 and C2 domains cannot be considered independent membrane binding modules.     

Study of the secondary structure of the C-terminal domain of the antiapoptotic protein bcl-2 and its interaction with model membranes

Bcl-2 is a protein which inhibits programmed cell death. It is associated to many cell membranes such as mitochondrial outer membrane, endoplasmic reticulum, and nuclear envelope, apparently through a C-terminal hydrophobic domain. We have used infrared spectroscopy to study the secondary structure of a synthetic peptide (a 23mer) with the same sequence as this C-terminal domain (residues 217-239) of Bcl-2. The spectrum of this peptide in D(2)O buffer shows an amide I' band with a maximum at 1622 cm(-1), which clearly indicates its tendency to aggregate in aqueous solvent. However, the peptide incorporated in multilamellar phosphatidylcholine membranes shows a totally different spectrum of the amide I' band, with a maximum at 1655 cm(-)(1), indicating a predominantly alpha-helical structure. Addition of the peptide to unilamellar vesicles destabilized them and released encapsulated carboxyfluorescein. Differential scanning calorimetry of dimyristoylphosphatidylcholine multilamellar vesicles in which the peptide was incorporated revealed that increasing concentrations of the peptide progressively broadened the pretransition and the main transition, as is to be expected for a membrane integral molecule. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in fluid phosphatidylcholine vesicles showed that increasing concentrations of the peptide produced increased polarization values, pointing to an increase in the apparent order of the membrane and indicating that high concentrations of the peptide considerably broaden the phase transition of dimyristoylphosphatidylcholine multilamellar vesicles. Quenching the intrinsic fluorescence of the Tyr-235 of the peptide, by KI, indicated that this aminoacyl residue is highly exposed to aqueous solvent when incorporated in phospholipid vesicles. The results are discussed in terms of their relevance to the proposed topology of insertion of Bcl-2 into biological membranes.     

The C1B domains of novel PKCε and PKCη have a higher membrane binding affinity than those of the also novel PKCδ and PKCθ

The C1 domains of novel PKCs mediate the diacylglycerol-dependent translocation of these enzymes. The four different C1B domains of novel PKCs (δ, ε, θ and η) were studied, together with different lipid mixtures containing acidic phospholipids and diacylglycerol or phorbol ester. The results show that either in the presence or in the absence of diacylglycerol, C1Bε and C1Bη exhibit a substantially higher propensity to bind to vesicles containing negatively charged phospholipids than C1Bδ and C1Bθ. The observed differences between the C1B domains of novel PKCs (in two groups of two each) were also evident in RBL-2H3 cells and it was found that, as with model membranes, in which C1Bε and C1Bη could be translocated to membranes by the addition of a soluble phosphatidic acid without diacylglycerol or phorbol ester, C1Bδ and C1Bθ were not translocated when soluble phosphatidic acid was added, and diacylglycerol was required to achieve a detectable binding to cell membranes. It is concluded that two different subfamilies of novel PKCs can be established with respect to their propensity to bind to the cell membrane and that these peculiarities in recognizing lipids may explain why these isoenzymes are specialized in responding to different triggering signals and bind to different cell membranes.     

The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).     

Interaction of the C2 domain from protein kinase C(epsilon) with model membranes

The C2 domain from protein kinase Cepsilon (PKCepsilon) binds to membranes but does not require Ca2+ to do so. This work examines the mode in which the conformation and organization of the phospholipids present in model membranes are altered by the presence of the C2 domain from PKCepsilon (C2-PKCepsilon). It is concluded from the results of differential scanning calorimetry that the protein shifted the temperature of the gel to the fluid phase transition of pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), widening the transition and increasing it to a higher temperature. When POPA was mixed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), the changes in the transition were smaller and no phase separation was observed. Experiments performed using magic angle spinning NMR showed that this C2 domain specifically affected POPA when the phospholipid was mixed with POPC, as indicated by the downfield shift in the isotropic resonance of POPA, the widening of the resonance peak, the decrease in T2, and the decrease in T1 observed at all temperatures. All these effects were quite marked compared with the very small effect observed with POPC, indicating the specificity of the effect. The presence of the C2-PKCepsilon protein changed the conformation of the polar head group of POPA, as shown by infrared spectroscopy. All these results clearly illustrate the electrostatic interaction that takes place between this C2 domain and membranes which contain POPA in the absence of Ca2+.