A chemoenzymatic synthesis of deoxy sugar esters involving stereoselective acetylation of hemiacetals catalyzed by CALB

An extension of the scope of the chemoenzymatic strategy for the synthesis of stereochemically pure pyranose deoxy sugar esters of different carboxylic acids has been achieved. The objective of the work was to extend the strategy to the synthesis of furanose deoxy sugar derivatives and additionally, to N-Boc-protected amino acid esters. With all used carboxylic acids (deoxycholic acid, α-methoxyphenylacetic acid, N-Boc-l-phenylalanine and N-Boc-l-tyrosine) the lipase-catalyzed stereoselective acetylation of furanose or pyranose hemiacetal moiety as a key step afforded one desired stereochemically pure acetylated hemiacetal deoxy sugar ester in high de.     

ABCA1 and scavenger receptor class B,type I,are modulators of reverse sterol transport at an in vitro blood-brain barrier constituted of porcine brain capillary endothelial cells

The objective of the present study was to investigate the involvement of key players in reverse cholesterol/24(S)OH-cholesterol transport in primary porcine brain capillary endothelial cells (pBCEC) that constitute the BBB. We identified that, in addition to scavenger receptor class B, type I (SR-BI), pBCEC express ABCA1 and apolipoprotein A-I (apoA-I) mRNA and protein. Studies on the regulation of ABCA1 by the liver X receptor agonist 24(S)OH-cholesterol revealed increased ABCA1 expression and apoA-I-dependent [3H]cholesterol efflux from pBCEC. In unpolarized pBCEC, high density lipoprotein, subclass 3 (HDL3)-dependent [3H]cholesterol efflux, was unaffected by 24(S)OH-cholesterol treatment but was enhanced 5-fold in SR-BI overexpressing pBCEC. Efflux of cellular 24(S)-[3H]OH-cholesterol was highly efficient, independent of ABCA1, and correlated with SR-BI expression. Polarized pBCEC were cultured on porous membrane filters that allow separate access to the apical and the basolateral compartment. Addition of cholesterol acceptors to the apical compartment resulted in preferential [3H]cholesterol efflux to the basolateral compartment. HDL3 was a better promoter of basolateral [3H]cholesterol efflux than lipid-free apoA-I. Basolateral pretreatment with 24(S)OH-cholesterol enhanced apoA-I-dependent basolateral cholesterol efflux up to 2-fold along with the induction of ABCA1 at the basolateral membrane. Secretion of apoA-I also occurred preferentially to the basolateral compartment, where the majority of apoA-I was recovered in an HDL-like density range. In contrast, 24(S)-[3H]OH-cholesterol was mobilized efficiently to the apical compartment of the in vitro BBB by HDL3, low density lipoprotein, and serum. These results suggest the existence of an autoregulatory mechanism for removal of potentially neurotoxic 24(S)OH-cholesterol. In conclusion, the apoA-I/ABCA1- and HDL/SR-BI-dependent pathways modulate polarized sterol mobilization at the BBB.     

Modification of low-density lipoprotein by myeloperoxidase-derived oxidants and reagent hypochlorous acid

Substantial evidence supports the notion that oxidative processes contribute to the pathogenesis of atherosclerosis and coronary heart disease. The nature of the oxidants that give rise to the elevated levels of oxidised lipids and proteins, and decreased levels of antioxidants, detected in human atherosclerotic lesions are, however, unclear, with multiple species having been invoked. Over the last few years, considerable data have been obtained in support of the hypothesis that oxidants generated by the heme enzyme myeloperoxidase play a key role in oxidation reactions in the artery wall. In this article, the evidence for a role of myeloperoxidase, and oxidants generated therefrom, in the modification of low-density lipoprotein, the major source of lipids in atherosclerotic lesions, is reviewed. Particular emphasis is placed on the reactions of the reactive species generated by this enzyme, the mechanisms and sites of damage, the role of modification of the different components of low-density lipoprotein, and the biological consequences of such oxidation on cell types present in the artery wall and in the circulation, respectively.     

An apparent decrease in cholesterol biosynthesis in peroxisomal-defective Chinese hamster ovary cells is related to impaired mitochondrial oxidation

Recent data suggest that impaired mitochondrial activities in Zellweger fibroblasts are related to defective peroxisome biogenesis and vice versa. To investigate the contribution of functional mitochondria to cholesterol biosynthesis, radioactive precursor molecules that form acetyl-CoA via beta-oxidation-independent (pyruvate) or -dependent (palmitate and octanoate) pathways were used. Production of both 14C-labeled cholesterol and 14C-labeled CO(2) from these radioactive tracers was significantly impaired in peroxisomal-defective ZR-82 Chinese hamster ovary cells in comparison to controls. In contrast, cholesterol synthesis from acetate--a tracer directly converted to acetyl-CoA without the involvement of mitochondrial activities--was threefold higher in ZR-82 cells than in controls. Pathways further contributing to cellular cholesterol homeostasis, i.e., receptor-mediated binding of exogenous lipoprotein-associated cholesterol as well as intracellular mobilization of cholesteryl ester deposits were similar in ZR-82 and controls. From these findings, we propose that peroxisomal dysfunction in ZR-82 cells is tightly coupled to impaired mitochondrial activities, e.g., defective mitochondrial beta-oxidation and formation of acetyl-CoA from short chain fatty acids resulting in a decreased rate of CO(2) production, and an apparent decrease in cholesterol biosynthesis. Actually, cholesterol biosynthesis from acetate is increased in the peroxisomal-defective cells. This explains previous conflicting conclusions.     

Characterization of an isoelectric focusing variant of SAA1 (ASP-72) in a family of Turkish origin.

An acidic variant of serum amyloid A (SAA) identified previously by isoelectrofocusing in a family of Turkish origin has been characterized at the genomic level. DNA sequence analysis revealed that individuals expressing the variant pI6.1/pI5.7 isoforms (the mother and three of four children) were heterozygous at the SAA1 gene locus. Their SAA1 gene sequences contained an adenine, as well as the usual guanine, at the position corresponding to the second base of codon 72. The presence of both bases predicts two SAA1 protein sequences, one having aspartic acid and the other glycine at position 72. While the Gly-72 SAA1 (+/- Arg-1) sequence represents the normal pI6.5/pI6.0 isoforms, the Asp-72 SAA1 (+/- Arg-1) sequence corresponds to the variant pI6.1/pI5.7 isoforms.     

The role of plasma phospholipid transfer protein (PLTP) in HDL remodeling in acute-phase patients

Lead toxicity is a major environmental health problem in the United States. Bone is the major reservoir for body lead. Although lead has been shown to impair bone metabolism in animals and at the cellular level, the effect of Pb(2+) at the molecular level is largely unknown. We have used circular dichroism (CD), and a hydroxyapatite binding assay to investigate the effect of Pb(2+) on the structure and mineral binding properties of osteocalcin, a noncollagenous bone protein. The CD data indicate Pb(2+) induces a similar structure in osteocalcin as Ca(2+) but at 2 orders of magnitude lower concentration. These results were explained by the more than 4 orders of magnitude tighter binding of Pb(2+) to osteocalcin (K(d)=0.085 microM) than Ca(2+) (K(d)=1.25 mM). The hydroxyapatite binding assays show that Pb(2+) causes an increased adsorption to hydroxyapatite, similar to Ca(2+), but at 2-3 orders of magnitude lower concentration. Low Pb(2+) levels (1 microM) in addition to physiological Ca(2+) levels (1 mM) caused a significant (40%) increase in the amount of mineral bound osteocalcin as compared to 1 mM Ca(2+) alone. These results suggest a molecular mechanism of Pb(2+) toxicity where low Pb(2+) levels can inappropriately perturb Ca(2+) regulated processes. In-vivo, the increased mineral bound osteocalcin could play a role in the observed low bone formation rates and decreased bone density observed in Pb(2+)-intoxicated animals.     

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Oxidation of LDL by the myeloperoxidase (MPO)-H₂O₂-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H₂O₂-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H₂O₂-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.     

Hypochlorite-modified low-density lipoprotein induces the apoptotic machinery in Jurkat T-cell lines

Myeloperoxidase is abundantly present in inflammatory diseases where activation of monocytes/macrophages and T-cell-mediated immune response occurs. The potent oxidant hypochlorous acid (HOCl), generated by the myeloperoxidase–H₂O₂–chloride system of activated phagocytes, converts low-density lipoprotein (LDL) into a proinflammatory lipoprotein particle. Here, we investigated the apoptotic effect of HOCl–LDL, an in vivo occurring LDL modification, on human T-cell lymphoblast-like Jurkat cells. Experiments revealed that HOCl–LDL, depending on the oxidant:lipoprotein molar ratio, induces apoptosis via activation of caspase-3, PARP cleavage and accumulation of reactive oxygen species. The absence of Fas-associated protein with death domain or caspase-8 in mutant cells did not prevent HOCl–LDL induced apoptosis. In contrast, overexpression of the anti-apoptotic Bcl-2 protein protects Jurkat cells against HOCl–LDL-induced apoptosis and prevents accumulation of reactive oxygen species. We conclude that HOCl–LDL-mediated apoptosis in Jurkat cells follows predominantly the intrinsic, mitochondrial pathway. Insitu experiments revealed that an antibody raised against HOCl–LDL recognized epitopes that colocalize both with myeloperoxidase and CD3-positive T-cells in human decidual tissue where local stimulation of the immune system occurs. We provide convincing evidence that formation of HOCl-modified (lipo)proteins generated by the myeloperoxidase–H₂O₂–chloride system contributes to apoptosis in T-cells.     

Mutant A53T alpha-synuclein induces neuronal death by increasing mitochondrial autophagy

Parkinson disease is characterized by the accumulation of aggregated α-synuclein as the major component of the Lewy bodies. α-Synuclein accumulation in turn leads to compensatory effects that may include the up-regulation of autophagy. Another common feature of Parkinson disease (PD) is mitochondrial dysfunction. Here, we provide evidence that the overactivation of autophagy may be a link that connects the intracellular accumulation of α-synuclein with mitochondrial dysfunction. We found that the activation of macroautophagy in primary cortical neurons that overexpress mutant A53T α-synuclein leads to massive mitochondrial destruction and loss, which is associated with a bioenergetic deficit and neuronal degeneration. No mitochondrial removal or net loss was observed when we suppressed the targeting of mitochondria to autophagosomes by silencing Parkin, overexpressing wild-type Mitofusin 2 and dominant negative Dynamin-related protein 1 or blocking autophagy by silencing autophagy-related genes. The inhibition of targeting mitochondria to autophagosomes or autophagy was also partially protective against mutant A53T α-synuclein-induced neuronal cell death. These data suggest that overactivated mitochondrial removal could be one of the contributing factors that leads to the mitochondrial loss observed in PD models.