Modulating reconstituted high density lipoprotein functionality to target the Pseudomonas aeruginosa quorum sensing system.

AIMS: The synthetic counterparts of serum high density lipoproteins (HDL; reconstituted HDL, reHDL) are assuming increasing importance as a therapeutic vector. They circulate not only in blood, but also outside the vascular compartment giving access to all body tissues. Presently, the therapeutic use of reHDL exploits inherent HDL functions. Our aim was to determine if HDL functionality could be modulated by attaching peptides not normally associated with the complex. MAIN METHODS: A peptide chimera was designed by linking the signal peptide of the HDL-associated enzyme paraoxonase-1 (PON1) to the coding region for the intracellular enzyme paraoxonase-2 (PON2). KEY FINDINGS: The signal peptide modified the properties of PON2, promoting its secretion from cells and binding to HDL. Enzyme activity of the chimera protein was highly stable. Conditioned HDL showed the functions of PON2 in its ability to hydrolyse typical PON2 substrates, namely homoserine lactones. Further in vitro studies showed that conditioned HDL was able to reduce the virulence of Pseudomonas aeruginosa. Both biofilm formation and the activation of the quorum sensing systems las and rhl, responsible for bacterial virulence, were significantly reduced. SIGNIFICANCE: The study provides proof of principal that the signal peptide of PON1 can be used to attach peptides to HDL and thus modulate HDL function. They may provide a vector that is ubiquitously distributed in extracellular body fluids for designing therapeutic strategies to address different pathophysiological states.

HDL-associated paraoxonase-1 can redistribute to cell membranes and influence sensitivity to oxidative stress.

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant capacity of the lipoprotein. In previous studies, we proposed that HDL promoted PON1 secretion by transfer of the enzyme from its plasma membrane location to HDL transiently anchored to the hepatocyte. This study examined whether PON1 can be transferred into cell membranes and retain its enzymatic activities and functions. Using Chinese hamster ovary and human endothelial cells, we found that recombinant PON1 as well as PON1 associated with purified human HDL was freely exchanged between the external medium and the cell membranes. Transferred PON1 was located in the external face of the plasma membrane of the cells in an enzymatically active form. The transfer of PON1 led to a gain of function by the target cells, as revealed by significantly reduced susceptibility to oxidative stress and significantly increased ability to neutralize the bacterial virulence agent 3-oxo-C(12)-homoserine lactone. The data demonstrate that PON1 is not a fixed component of HDL and suggest that the enzyme could also exert its protective functions outside the lipoprotein environment. The observations may be of relevance to tissues exposed to oxidative stress and/or bacterial infection.

Paraoxonase-1 promoter polymorphism C--107T and serum apolipoprotein AI interact to modulate serum paraoxonase-1 status.

OBJECTIVES: The objective was to examine the hypothesis that modifications to paraoxonase-1 specific activity (SP, activity per unit mass peptide) could contribute to serum paraoxonase-1 status, a determinant of the clinical efficacy of the enzyme. METHODS: Enzyme activities and concentrations were determined in a large population (n=912) of patients and controls. SP were subsequently examined as a function of paraoxonase-1 gene polymorphisms, plasma lipids and lipoproteins, and physiological and pathophysiological parameters. RESULTS: Pathophysiological parameters (diabetes, metabolic syndrome, smoking, aging) did not promote variations in paraoxonase-1 SP, whilst coronary disease lowered SP (P<0.003). No serum lipid, apolipoprotein or lipoprotein component had an impact on specific activity, with the exception of apolipoprotein AI (P<0.005, both substrates). The paraoxonase-1 promoter C--107T and Q192R polymorphisms influenced SP and, together with apolipoprotein AI, were highly significant, independent determinants in regression models. There was an interaction between apolipoprotein AI and the C--107T polymorphism, which significantly modulated SP and serum paraoxonase-1 status. CONCLUSIONS: Enzyme inactivation giving rise to modulated activity per unit mass of peptide is not a major contributor to pathological effects of disease on serum paraoxonase-1 status. The C--107T polymorphism and serum apolipoprotein AI have major impacts individually on SP and also provide an example of gene-environment interaction to modulate such activities. These effects accentuate the differences between--107C and--107T allele carriers in terms of serum paraoxonase-1 status. The data underline the complexity of the factors that determine serum paraoxonase-1 status and suggest that the latter would benefit from therapeutic modulation of serum high density lipoproteins.