Oscar A. Bizzozero

Elevated protein carbonylation in the brain white matter and gray matter of patients with multiple sclerosis

Oxidative stress has been implicated in the pathophysiology of multiple sclerosis (MS). Increased levels of reactive oxygen species (ROS) derived from infiltrating macrophages and microglial cells have been shown to reduce the levels of endogenous antioxidants and to cause the oxidation of various substrates within the MS plaque. To determine whether oxidative damage takes place beyond visible MS plaques, the occurrence of total carbonyls (TCOs) and protein carbonyls (PCOs) in the normal‐appearing white matter (NAWM) and gray matter (NAGM) of eight MS brains was assessed and compared with those of four control brains. The data show that most (7/8) of the MS‐WM samples contain increased amounts of PCOs as determined by reaction with 2,4‐dinitrophenylhydrazine and Western blot analysis. These samples also have high levels of glial fibrilary acidic protein (GFAP), suggesting that oxidative damage is related to the presence of small lesions. In contrast, we detected no evidence of protein thiolation (glutathionylation and cysteinylation) in the diseased tissue. To our surprise, MS‐NAGM specimens with high GFAP content also showed three times the concentration of TCOs and PCOs as the controls. The increase in PCOs is likely to be a consequence of reduced levels of antioxidants, in that the concentration of nonprotein thiols in both MS‐WM and ‐GM decreased by 30%. Overall, our data support the current view that both NAWM and ‐GM from MS brains contain considerable biochemical alterations. The involvement of GM in MS was also supported by the decrease in the levels of neurofilament light protein in all the specimens analyzed. To the best of our knowledge, this is the first study demonstrating the presence of increased protein carbonylation in post‐mortem WM and GM tissue of MS patients.

Cytoskeletal protein carbonylation and degradation in experimental autoimmune encephalomyelitis

Protein carbonylation, the non‐enzymatic addition of aldehydes or ketones to specific amino acid residues, has been implicated in the pathophysiology of multiple sclerosis. In this study, we investigated whether protein carbonyls also accumulate in the spinal cord of Lewis rats with acute experimental autoimmune encephalomyelitis (EAE). Western blots analysis after derivatization with dinitrophenyl hydrazine (oxyblot) showed elevated protein carbonylation at the time of maximal clinical disability. During the same period glutathione levels were substantially reduced, suggesting a causal relationship between these two markers. In contrast, lipid peroxidation products accumulated in EAE spinal cord well before the appearance of neurological symptoms. Carbonyl staining was not restricted to inflammatory lesions but present throughout the spinal cord particularly in neuronal cell bodies and axons. By 2‐dimensional‐oxyblot, we identified several cytoskeletal proteins, including β‐actin, glial acidic fibrillary protein, and the neurofilament proteins as the major targets of carbonylation. These findings were confirmed by pull‐down experiments, which also showed an increase in the number of carbonylated β‐actin molecules and a decrease in that of oxidized neurofilament proteins in EAE. These data suggest the possibility that oxidation targets neurofilament proteins for degradation, which may contribute to axonal pathology observed in multiple sclerosis and EAE.

Fatty Acid Composition of Human Myelin Proteolipid Protein in Peroxisomal Disorders

Abstract: Myelin proteolipid protein (PLP) is an acylated protein which contains approximately 2 mol of ester‐bound fatty acids. In this study, the amount and composition of fatty acids covalently bound to human myelin PLP were determined during development and in peroxisomal disorders. Palmitic, oleic, and stearic acids accounted for most of the PLP acyl chains. However, in contrast to PLP in other species, human PLP contains relatively more very long chain fatty acids (VLCFA). The fatty acid composition remained essentially unchanged between 1 day and 74 years of age. The total amount of fatty acid bound to PLP was not altered in any of the pathological cases examined. However, in the peroxisomal disorder adrenoleukodystrophy, the proportions of saturated and, to a lesser extent, monounsaturated VLCFA bound to PLP were increased at the expense of oleic acid. Smaller, but significant, changes were observed in adrenomyeloneuropathy. The reduction in the levels of oleic acid was also observed in two other peroxisomal disorders, the cerebrohepatorenal (Zellweger) syndrome and neonatal adrenoleukodystrophy, as well as in the lysosomal disorder Krabbe globoid cell leukodystrophy. However, in these disorders, the decrease in oleic acid occurred at the expense of stearic acid, and not VLCFA. The results indicate that, although a characteristic PLP fatty acid pattern is normally maintained, changes in the acyl chain pool can ultimately be reflected in the fatty acid composition of the protein. The altered PLP‐acyl chain pattern in peroxisomal disorders may contribute to the pathophysiology of these devastating disorders.

Structural determinants influencing the reaction of cysteine-containing peptides with palmitoyl-coenzyme A and other thioesters

Non-enzymatic thioesterification of specific cysteinyl peptides with fatty acyl-CoA has been previously demonstrated in both liposomes and aqueous medium. To identify the molecular basis for the differential reactivity of polypeptides in aqueous solutions, 26 synthetic cysteinyl peptides encompassing the palmitoylation sites of well known proteins (protein zero, proteolipid protein, beta-adrenergic receptor, p21(K-ras), transferrin receptor, CD-4 and SNAP-25) and six small thiol compounds were incubated separately with [3H]palmitoyl-CoA, [14C]acetyl-CoA and p-nitrophenyl thioacetate (NPTA). For each peptide, both the observed reaction rate constant at pH 7.5 and the pH-independent rate constant (k(2)) were calculated, and reactivity of the attacking sulfhydryl group was characterized using the Brønsted equation (log k(2)=beta(nuc) pK(a)+C). In general, peptides bearing basic and aromatic amino acid residues showed the lowest thiol pK(a)s, and consequently displayed the highest acylation rates. Reaction with palmitoyl-CoA was complicated to analyze because of the variable partition of peptides in the acyl chain donor/detergent micelles. In contrast, a linear Brønsted relationship was found for the reaction of the peptides with the water-soluble acetyl-CoA (beta(nuc)=0.59). A similar beta(nuc) value was obtained with the neutral NPTA, indicating that electronic effects other than those responsible for the acid-base properties of the thiol are less important. Thus, the concentration of the thiolate anion appears to be the major factor influencing the rate of the nucleophilic substitution reaction. These findings and the fact that the acylation sites in most proteins are surrounded by basic amino acids may partially explain the specificity of non-enzymatic palmitoylation regarding the acceptor sequences.

Accumulation of protein carbonyls within cerebellar astrocytes in murine experimental autoimmune encephalomyelitis

Recent work from our laboratory has implicated protein carbonylation in the pathophysiology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The present study was designed to determine the changes in protein carbonylation during disease progression and to identify the target cells and modified proteins in the cerebellum of EAE animals, prepared by active immunization of C57/BL6 mice with MOG35–55 peptide. In this model, protein carbonylation was maximal at the peak of the disease (acute phase), to decrease thereafter (chronic phase). Double‐immunofluorescence microscopy of affected cerebella showed that carbonyls accumulate in white matter astrocytes and to a lesser extent in microglia/macrophages, in both the acute and the chronic phase. Surprisingly, T cells, oligodendrocytes, and neurons were barely stained. By 2D oxyblot and mass spectrometry, β‐actin, β‐tubulin, GFAP, and HSC‐71 were identified as the major targets of carbonylation throughout the disease. Using a pull‐down/Western blot method, we found a significant increase in the proportion of carbonylated β‐actin, β‐tubulin, and GFAP in the chronic phase but not in the acute phase. These results suggest that as disease progresses from the inflammatory to the neurodegenerative phase there may be an inappropriate removal of oxidized cytoskeletal proteins. Additionally, the extensive accumulation of carbonylated GFAP in the chronic phase of EAE may be responsible for the abnormal shape of astrocytes observed at this stage.

Evidence of Nitrosative Damage in the Brain White Matter of Patients with Multiple Sclerosis

Nitric oxide (NO) has been implicated in the pathophysiology of both experimental autoimmune encephalomyelitis and multiple sclerosis (MS). NO-mediated protein damage in MS appears to be confined to large plaques where 3-nitrotyrosine has been detected. To determine whether nitrosative damage takes place beyond visible MS plaques, the occurrence of various NO-triggered protein modifications in normal-appearing white matter (NAWM) of eight MS brains was assessed and compared to that in white matter (WM) of four control brains. As determined by amino acid analysis and western blotting, no evidence of tyrosine nitration was found in the MS samples studied, suggesting that they did not contain appreciable amounts of plaque-derived material. The amino acid composition of total myelin proteins and proteolipid protein (PLP) was also unaltered in the diseased tissue, as was the fatty acid composition of PLP. In addition, we detected no changes in the number of protein free thiols suggesting that oxidation do not occur to any appreciable extent. However, the levels of nitrite in MS-NAWM were higher than those in control WM, while in the MS-gray matter (GM) the concentration of this ion was unaltered. Furthermore, five of the MS samples analyzed, and the same as those with high levels of glial fibrilary acidic protein, showed increased amounts of protein nitrosothiols as determined by the “biotin switch” method. S-nitrosation of GM proteins was again normal. There was no indication of N-nitrosation of tryptophan and N-terminal amino groups in both control and MS tissue. Overall, the data suggests that WM, but not GM, from MS brains is subjected to considerable nitrosative stress. This is the first report to present direct evidence of increased protein S-nitrosation and nitrite content in the brain parenchyma of MS patients.

Myelin P0 Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated

Abstract: P0, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys153. To gain insights into the mechanism of P0 acylation, the in vitro palmitoylation of both P0 and a synthetic Cys153‐containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P0 with [3H]palmitoyl‐CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P0 was not affected by boiling the isolated P0 for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonezymatic nature of the reaction. After chemical deacylation, P0 was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [14C]palmitoyl‐CoA, the synthetic peptide encompassing the natural P0 acylation site (I150RYCWLRR157) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37°C, peptide palmitoylation followed a second‐order reaction (k2 = 246 ± 6 M−1 min−1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl‐CoA. The activation energy calculated from the Arrhenius plot is ∼2 kcal/mol and much lower than that of enzyme‐catalyzed transacylations. Finally, two other P0 peptides (V121PTRYG126 and K109TSQVTL115) as well as various unrelated thiol‐containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.

Chemical deacylation reduces the adhesive properties of proteolipid protein and leads to decompaction of the myelin sheath

Myelin proteolipid protein (PLP) contains thioester‐bound, long‐chain fatty acids which are known to influence the structure of the molecule. To gain further insights into the role of this post‐translational modification, we studied the effect that chemical deacylation of PLP had on the morphology of myelin and on the proteins ability to mediate the clustering of lipid vesicles. Incubation of rat optic nerves in isoosmotic solutions containing 100 mm hydroxylamine (HA) pH 7.4 led to deacylation of PLP and decompaction of myelin lamellae at the level of the intraperiod line. Incubation of nerves with milder nucleophilic agents (Tris and methylamine) or diluted HA, conditions that do not remove protein‐bound fatty acids, caused no alterations in myelin structure. Other possible effects of HA which could have affected myelin compaction indirectly were ruled out. Incubation of optic nerves with 50 mm dithioerythritol (DTE) also led to the splitting of the myelin intraperiod line and this change again coincided with the removal of fatty acids. In addition, the apparently compacted CNS myelin in the PLP‐less myelin‐deficient rat, like that in tissue containing deacylated PLP, was readily decompacted upon incubation in isoosmotic buffers, suggesting that the function of PLP as a stabilizer of the interlamellar attachment is, at least in part, mediated by fatty acylation. Furthermore, in contrast to the native protein, PLP deacylated with either HA or DTE failed to induce the clustering of phosphatidylcholine/cholesterol vesicles in vitro. This phenomenon is not due to side‐effects of the deacylation procedure since, upon partial repalmitoylation, the protein recovered most of its original vesicle‐clustering activity. Collectively, these findings suggest that palmitoylation, by influencing the adhesive properties of PLP, is important for stabilizing the multilamellar structure of myelin.

Fatty acid acylation of rat brain myelin proteolipid protein in vitro: identification of the lipid donor.

Abstract: The immediate acyl chain donor for fatty acid esterification of proteolipid protein (PLP) was identified in an in vitro system. Rat brain total membranes, after removal of crude nuclear and mitochondrial fractions, were incubated with radioactive acyl donors, extracted with chlorofrm/methanol, and analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. In the presence of [3H]palmitic acid, CoA, ATP, and Mg2+, acylation of endogenous PLP occurred at a linear rate for at least 2 h. The radioactivity was associated with the protein via an ester linkage, mainly as palmitic acid. Omission of ATP, CoA, Mg2+, or all three reduced fatty acid incorporation into PLP to 44, 27, 8, and 4%, respectively, of the values in the complete system. Incubation of the membrane fraction with [3H]palmitoyl‐CoA in the absence of CoA and ATP led to highly labeled PLP. These data demonstrate that activation of free fatty acid is required for acylation. Phospholipids and glycolipids were not able to acylate the PLP directly. Finally, when isolated myelin was incubated with [3H]palmitoyl‐CoA in the absence of cofactors, only PLP was labeled, thus confirming the identity of palmitoyl‐CoA as the direct acyl chain donor and suggesting that the acylating activity and the PLP pool available for acylation are both in the myelin.

Intracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cord

Protein S‐nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory and neurodegenerative disorders. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S‐NO linkage in intact cells. To address this issue, we conducted chase experiments in spinal cord slices incubated with S‐nitrosoglutathione (GSNO). The results show that removal of GSNO leads to a rapid disappearance of PrSNOs (t½ ∼ 2 hr), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester. Moreover, PrSNOs are stable in the presence of the GSH depletor diethyl maleate, indicating that GSH is critical for protein denitrosylation. Inhibition of GSH‐dependent enzymes (glutathione S‐transferase, glutathione peroxidase, and glutaredoxin) and enzymes that could mediate denitrosylation (alcohol dehydrogense‐III, thioredoxin and protein disulfide isomerase) do not alter the rate of PrSNO decomposition. These findings and the lack of protein glutathionylation during the chase indicate that most proteins are denitrosylated via rapid transnitrosylation with GSH. The differences in the denitrosylation rate of individual proteins suggest the existence of additional structural factors in this process. This study is relevant to our recent discovery that PrSNOs accumulate in the central nervous system of patients with multiple sclerosis.