The role of osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand in human microvascular endothelial cell survival.

Endothelial cell survival and antiapoptotic pathways, including those stimulated by extracellular matrix, are critical regulators of vasculogenesis, angiogenesis, endothelial repair, and shear-stress-induced endothelial activation. One of these pathways is mediated by alpha(v)beta(3) integrin ligation, downstream activation of nuclear factor-kappaB, and subsequent up-regulation of osteoprotegerin (OPG). In this study, the mechanism by which OPG protects endothelial cells from death was examined. Serum-starved human microvascular endothelial cells (HMECs) plated on the alpha(v)beta(3) ligand osteopontin were protected from cell death. Immunoprecipitation experiments indicated that OPG formed a complex with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in HMECs under these conditions. Furthermore, inhibitors of TRAIL, including recombinant soluble TRAIL receptors and a neutralizing antibody against TRAIL, blocked apoptosis of serum-starved HMECs plated on the nonintegrin attachment factor poly-d-lysine. Whereas TRAIL was unable to induce apoptosis in HMECs plated on osteopontin, the addition of recombinant TRAIL did increase the percentage of apoptotic HMECs plated on poly-d-lysine. This evidence indicates that OPG blocks endothelial cell apoptosis through binding TRAIL and preventing its interaction with death-inducing TRAIL-receptors

Peptidylarginine deiminase: a candidate factor in demyelinating disease.

In earlier studies we demonstrated that an increase in the relative amounts of citrullinated myelin basic protein (MBP) was found in multiple sclerosis (Moscarello et al. 1994). To determine the temporal relationship between the citrullinated MBP and peptidylarginine deiminase (PAD), the enzyme responsible for deiminating arginyl residues in proteins, we studied enzyme activity, enzyme protein, PAD mRNA in a spontaneously demyelinating transgenic mouse model and we correlated the amount of PAD with citrullinated MBP. Both PAD protein as measured in an immunoslot blot method and PAD RNA were elevated. In fractionation studies we showed that the increase in PAD enzyme was due to an increase in the PAD found in membrane fractions and not the soluble PAD (PADII). From our data we concluded that up-regulation of myelin-associated PAD was responsible for the increase in citrullinated MBP in our transgenic mice prior to onset of clinical or pathological signs of demyelination. We postulate that a similar mechanism may be responsible for the increase in citrullinated MBP in multiple sclerosis.

Deimination of myelin basic protein. 1. Effect of deimination of arginyl residues of myelin basic protein on its structure and susceptibility to digestion by cathepsin D.

The effect of deimination of arginyl residues in bovine myelin basic protein (MBP) on its susceptibility to digestion by cathepsin D has been studied. Using bovine component 1 (C-1) of MBP, the most unmodified of the components with all 18 arginyl residues intact, we have generated a number of citrullinated forms by treatment of the protein with purified peptidylarginine deiminase (PAD) in vitro. We obtained species containing 0-9.9 mol of citrulline/mol of MBP. These various species were digested with cathepsin D, a metalloproteinase which cleaves proteins at Phe-Phe linkages. The rate of digestion compared to component 1 was only slightly affected when 2.7 or 3.8 mol of citrulline/mol of MBP was present. With 7.0 mol of citrulline/mol of MBP, a large increase in the rate of digestion occurred. No further increase was observed with 9.9 mol of citrulline/mol of MBP. The immunodominant peptide 43-88 (bovine sequence) was released slowly when 2.7 and 3.8 mol of citrulline/mol of MBP was present, but it was released rapidly when 7.0 mol of citrulline/mol of MBP was present. The dramatic change in digestion with 7.0 mol of citrulline/mol of MBP or more could be explained by a change in three-dimensional structure. Molecular dynamics simulation showed that increasing the number of citrullinyl residues above 7 mol/mol of MBP generated a more open structure, consistent with experimental observations in the literature. We conclude that PAD, which deiminates arginyl residues in proteins, decreases both the charge and compact structure of MBP. This structural change allows better access of the Phe-Phe linkages to cathepsin D. This scheme represents an effective way of generating the immunodominant peptide which sensitizes T-cells for the autoimmune response in demyelinating disease.

Deimination of myelin basic protein. 2. Effect of methylation of MBP on its deimination by peptidylarginine deiminase.

Deimination of myelin basic protein (MBP) has been implicated in the chemical pathogenesis of multiple sclerosis (MS). Degradation of bovine MBP by cathepsin D, a myelin-associated protease, was increased when 6 arginyl residues were deiminated and became very rapid when all 18 arginyl residues were deiminated. Since MBP contains a number of modifications, including methylation, phosphorylation, etc., we studied the effect of methylation, an irreversible modification, to determine how this modification affected deimination. Methylation of Arg 106 in bovine MBP (Arg 107 in human), a naturally occurring modification of MBP, has been shown to affect the deimination of arginyl residues in the present study. Since fractionation of MBP into unmethylated, monomethylated, and dimethylated species cannot be done readily on a preparative scale, mass spectrometry with the Q-TOF instrument resolved these species readily since each differed from the other by 14 atomic mass units (amu). Examination of five different hMBP samples, two from normal brain and three from MS brain, revealed that increased deimination of arginyl residues correlated with a decreased methylation of Arg 107 (human sequence). To study this process in vitro, bovine MBP (bMBP) was used. Component 1 (C-1) is the most cationic of the MBP "charge isomers" and the most unmodified, in which all arginyl residues are intact. It was deiminated to various extents with purified bovine brain peptidylarginine deiminase, generating a number of species containing 0-13.7 mol of citrulline/mol of bMBP. Mass spectrometry of each of these species permitted us to determine the influence of methylation of Arg 106 (bovine sequence) on deimination by this enzyme. We found that bMBP with unmethylated arginine was deiminated at a rate of 0.081 mol of citrulline/min, with monomethylarginine, 0.068 mol of citrulline/min, and with dimethylarginine, 0.036 mol of citrulline/min. We suggest that the methylated arginyl residue becomes sequestered in the hydrophobic beta-sheet structure and disrupts the three-dimensional structure of the protein so that other arginyl residues are less accessible to peptidylarginine deiminase.

The developmental expression and activity of peptidylarginine deiminase in the mouse.

We have measured the expression and activity of peptidylarginine deiminase (PAD, EC 3.5.3.15), the enzyme responsible for converting arginyl residues in proteins to citrullines, in normal mouse brain homogenate. PAD transcripts were detected as early as five days and were maximal at one month of age. The enzyme protein was also detected at 5 days in an antibody dependent assay and was maximal at 2 months of age, 1 month later than the maximum expression of transcripts. As expected, enzyme activity had a similar developmental profile to that of the enzyme protein. In isolated mouse brain compact myelin, the activity was highest at 15 days and fell rapidly to 15% of this level by 1-2 months. In the 'loose' myelin fraction (heavy myelin) it remained at the same high level form from 15 days to 8 months. The activity in compact myelin was about 15 times greater than the activity in brain homogenate, suggesting much of the enzyme was localized to myelin.

A novel microtubule independent effect of paclitaxel: the inhibition of peptidylarginine deiminase from bovine brain.

Although the principal effect of paclitaxel (taxol) is in preventing depolymerization of microtubules, other effects have been described recently. In the present manuscript, we demonstrate an inhibitory effect on the enzyme peptidylarginine deiminase (PAD) which converts peptidyl bound arginine to citrulline. To study the mechanism of action of the drug on PAD, a number of studies were carried out with purified enzyme. With the synthetic substrate benzoyl-arginine ethyl ester (BAEE), almost total inhibition of activity was observed at 12. 5 mM. With myelin basic protein (MBP) as a substrate, deimination of arginyl residues was prevented by 0.5 mM paclitaxel. The velocity-substrate curve was unusual since substrate enhancement was observed at 5 mM BAEE. These data suggested the presence of two binding sites on the enzyme. Inhibition of activity by paclitaxel was non-competitive for both sites.

Do lipid lowering drugs reduce the risk of coronary heart disease?

Historically, a wide range of drugs have been used to treat hyperlipidemias. These include fibrates whose main action is to lower plasma triglycerides; bile-acid sequestering resins introduced to reduce plasma cholesterol; and more recently a family of statins designed to inhibit the rate-limiting enzyme of cholesterol biosynthesis, HMG-CoA reductase. Early trials employing rather small numbers of subjects with established coronary artery disease (CAD) demonstrated that lipid reduction was often but not always associated with a lowered incidence of clinical end-points, including death and non-fatal myocardial infarction. However, significant angiographic benefit was rarely demonstrated in these investigations. More recent trials based on statins have shown a reduction in clinical events not only in patients with CAD, but also in healthy subjects given these drugs for primary prevention. Differences in design, duration, the role of confounders such as risk factors other than lipids, and the frequently poor correlation between angiographic changes and clinical outcomes are discussed.

Acylation of myelin basic protein peptide 1-21 with alkyl carboxylates 2-10 carbons long affects secondary structure and posttranslational modification.

A peptide consisting of the first 21 residues of human myelin basic protein (MBP) was synthesized. The N-terminal alanine of portions was blocked in separate experiments with alkyl carboxylates varying in size from 2 to 10 carbon atoms. The effects of these different alkyl carboxylates at the N-terminus on the secondary structure was studied by circular dichroism (250-190 nm). In water, the spectra of the unblocked peptide suggested unordered structure with large negative ellipticities at 198 nm. Addition of an acetyl group altered the magnitude of [theta]198 from -21856 +/- 2319 to -11095 +/- 1000 deg cm2 dmol-1, suggesting a significant increase in ordered structure. When peptides with longer alkyl carboxylates, acylated at the N-termini, were studied, the magnitude of theta 198 approached that of the unblocked peptide but greater negative ellipticities were observed for the C8 and C10 alkyl carboxylates. The theta 222 values were generally low (-1803 +/- 463) but increased with increasing length of the alkyl carboxylate to about -3200 deg cm2 dmol-1, suggesting that little alpha-helical structure was present. The spectra were also taken in lipid-mimetic solvents, including 2-propanol, methanol, and lysophosphatidylglycerol (lysoPG). In general the theta 198 and theta 222 values were suggestive of increased structure in these environments compared to water. In 90% 2-propanol the theta 198 of the unblocked peptide did not change when an acetyl group was added to the N-terminus (9088 compared to 8477 deg cm2 dmol-1). Addition of longer alkyl carboxylates correlated with larger, negative ellipticities.(ABSTRACT TRUNCATED AT 250 WORDS)