The synthesis and analysis of S-nitorsylated paraoxonase 1.

Post-translational modification (PTM) of proteins plays a crucial role in health and disease by affecting numerous aspects of protein structure, function, stability and subcellular localization. Protein S-nitrosylation is one type of PTM that involves the covalent modification of cysteine sulfhydryl groups with nitric oxide (NO) and has a regulatory impact similar to phosphorylation. The enzyme paraoxonase 1 (PON1) is associated with high-density lipoprotein (HDL), and is responsible for many of HDL's antiatherogenic properties. The enzyme contains a free thiol group at Cys-284 which can also be modified covalently. As part of our effort to study the effect of PTMs on PON1 activities and properties and its implication for cardiovascular disease, we examined PON1's ability to undergo S-nitrosylation on its free Cys-284. Recombinant (re) PON1 was trans-S-nitrosylated by several NO donors, glutathione-NO (GSNO) was found to be the most effective. The S-nitrosylated rePON1 was analyzed by Q-TOF LC/MS and by Saville-Griess assay: the two analytical methods revealed closely similar results. rePON1 was also nitrosylated by nitrosylated human serum albumin (HSA-NO) via protein-protein trans-nitrosylation. HSA-NO transferred an NO group to rePON1 much more efficiently than GSNO with the formation of a higher than 70% rePON-NO when incubated with a 40-fold excess of a HSA-NO/HSA mixture. RePON1-NO was relatively stable: storage for 3days at 37°C resulted in only 25% decomposition. This is the first report of PON1's S-nitrosylation via GSNO and HSA-NO.

Human carotid plaque phosphatidylcholine specifically interacts with paraoxonase 1, increases its activity, and enhances its uptake by macrophage at the expense of its binding to HDL.

Human carotid atherosclerotic plaque is in direct contact with circulatory blood components. Thus, plaque and blood components may affect each other. The current study presents the effects of plaque chloroform:methanol (C:M) extract on the HDL-associated enzyme paraoxnase 1 (PON1). This study is part of our investigation on the mutual effects of the interactions between atherosclerotic lesions and blood components. Recombinant PON1 (rePON1) was incubated with the human carotid plaques C:M extract and PON1 activities were analyzed. Lactonase and paraoxonase activities were elevated due to C:M treatment, by 140 and by 69%, respectively. Analytical chemistry analyses revealed specific phosphatidylcholines (PCs) as the plaque active components. Tryptophan fluorescence quenching assay, together with molecular docking, shows that PON1 activity is enhanced in correlation with the level of PC affinity to PON1. Molecular docking revealed that PCs interact specifically with H2-PON1 α-helix, which together with H1 enzyme α-helix links the protein to the HDL surface. These findings are supported by additional results from the PON1 ∆20 mutant that lack its H1-α-helix. Incubation of this mutant with the plaque C:M extract increased PON1 activity by only 20%, much less than the wild-type PON1 that elevated PON1 activity at the same concentration by as much as 95%. Furthermore, as much as the affinity of the enzyme to the PC was augmented, the ability of PON1 to bind to the HDL particle decreased. Finally, PON1 interaction with PC enhance its uptake into the macrophage cytoplasm. In conclusions, Specific lesion phosphatidylcholines (PCs) present in the human carotid plaque significantly enhance PON1 catalytic activities due to their interaction with the enzyme. Such a lesion׳s PC-PON1 interaction, in turn, competes with HDL PCs and enhances PON1 uptake by macrophage at the expense of PON1 binding to the HDL.

The impact of PEGylation on protein immunogenicity.

Covalent attachment of PEG (PEGylation) is widely used to improve the pharmaceutical properties of therapeutic proteins. The applicability and safety of this method have been proven by the use of various PEGylated pharmaceutical proteins approved by the Food and Drug Administration (FDA). One of the properties attributed to PEGylation is immunogenicity reduction of the PEGylated protein. In this study, the impact of PEGylation on immunogenicity was tested and compared for two proteins (chicken IgY and horse IgG) in two strains of mice (Balb/c and C57BL/6) for two routes of administration (i.v. and i.m.) and two sizes of PEG (5 kD and 20 kD). The influence of PEG was shown to be inconsistent between the mouse strains and routes of administration, even with the same tested protein. Consequently, immunogenicity reduction by PEGylation cannot be predicted or assumed; it must be tested on an individual case basis.