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PON1/APO A-1 mimetic peptides

Written by Le Tien Dung on .

Nguyen Duy Su, called as Su, is a friend of mine during my study in South Korea. He was an MS and then a PhD student at Chungnam National University. His work has been focused intensively on the peptides that have antioxidant activities through interaction with lipoproteins.

Su got his Bachelor of Pharmacy at the Hanoi College of Pharmacy and earned his MS from Chungnam National University (South Korea). He has successfully defended his PhD thesis and will have his PhD degree awarded by the same University within the next few months. Su works really hard and accomplished several papers during his graduate study. It is our pleasure to have his Thesis abstract published here. The thesis entitled "ANTIOXIDANT ACTIONS OF PON1/APO A-I MIMETIC PEPTIDES AND THEIR INTERACTIONS WITH LIPOPROTEINS". A completed list of Su's publications is available here. Su likes to talk about science and research, we talked alots about experiments and researchs each time we met. Though he has not got his degree, Su has got several offers for postdoc training, including offers from US and EU.

ANTIOXIDANT ACTIONS OF PON1/APO A-I MIMETIC PEPTIDES AND THEIR INTERACTIONS WITH LIPOPROTEINS 

The studies presented in this dissertation have been targeted to improvement of HDL quality as a therapeutic strategy for cardiovascular diseases. To accomplish this objective, we have endeavored to pinpoint and design the antioxidant mimetic peptides of paraoxonase1 (PON1) and apolipoprotein A-I (Apo A-I), two HDL-associated proteins mainly responsible for multiple beneficial actions of HDL.  The content of this dissertation comprises of four parts 

1. Overview of atherosclerosis and strategies for development of therapeutic targets in atherosclerosis

We attempted to give an overview of atherosclerosis and how research in this field has progressed over the past years. In addition, the molecular and cellular biology as well as functional aspects of lipoproteins involved in atherosclerosis are also profoundly reviewed. Especially, the practical strategies for development of therapeutic targets in atherosclerosis are highlighted, with a particular emphasis on refinement of HDL quality.{mosgoogle}

2. Regulation of PON1 activities by phospholipidsThis part focuses on delineating the structure and physiological functions of paraoxonase1, in an antioxidant protein associated with HDL, and the relationship between its structure and function are discussed. In addition, we extensively investigated the potential roles of various phospholipids in regulating PON1 activities. For this purpose, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length–dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (≥0.1 mM) inhibited both activities. Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity. 

3. Broad-spectrum antioxidant peptides derived from His residue-containing sequences present in PON1

Given the importantly antioxidant properties of PON1, we wished to identify mimetic peptides present in PON molecule and investigated the antioxidant activity by employing various antioxidant assay. We examined Cu2+-binding property of paraoxonase 1 (PON1), and antioxidant actions of peptides, resembling His residue-containing sequences in PON1, against oxidations by Cu2+, peroxyl radicals or HOCl. When Cu2+-binding property of PON1 was examined spectrophotometrically, the maximal Cu2+ binding was achieved at 1:1 molar ratio of PON1: Cu2+. Additionally, Cu2+-catalyzed oxidative inactivation of PON1 was prevented by Ca2+-depleted PON1  at 1:1 ratio, but not diethylpyrocarbonate-modified PON1, suggesting the participation of His residue in Cu2+-binding. When His-containing peptides were examined for antioxidant actions, those with either His residue at N-terminal position 2 or 3, or His-Pro sequence at C-terminal remarkably prevented Cu2+-mediated LDL oxidation and PON1 inactivation. Especially, FHKALY, FHKY or NHP efficiently prevented Cu2+-induced LDL oxidation (24 hr), indicating a tight binding of Cu2+ by peptides. In support of this, the peptide: Cu2+ complexes exhibited a superoxide-scavenging activity. Separately, in oxidations by 2, 2’-azobis-2-amidinopropane hydrochloride or HOCl, the presence of Tyr or Cys residue markedly enhanced antioxidant action of His-containing peptides. These results indicate that His-containing peptides with Tys or Cys residues correspond to broad spectrum antioxidants in oxidation models employing Cu2+, AAPH or HOCl.

4. Apolipoprotein A-I-mimetic peptides with antioxidant actions

The purpose of this part is to design and synthesize a number of Apo A-I-derived peptides, and to examine their biological actions as well as their interaction with lipid as well as PON molecules. To augment antioxidant action of apolipoprotein A-I (Apo A-I)-mimetic peptide, the peptide F3,6,14,1818A (DWFKAFYDKVAEKFKEAF) was modified by incorporating antioxidant amino acid residues. Introduction of His residue at position 2 or 3 at N-terminal of the peptide remarkably enhanced antioxidant action against Cu2+ oxidation of LDL and the capability of sequestering Cu2+. Likewise, the substitution of Ala for Cys residue at position 12 increased antioxidant action against Cu2+ oxidation of LDL. Additionally, the Cys substitution contributed to enhanced capabilities in the removal of hypochlorous acid (HOCl) and 13-hydroperoxyoctadecadienoic acid. Furthermore, the combined incorporation of His and Cys residues enhanced antioxidant actions in preventing Cu2+ oxidation and reducing HOCl and hydroperoxide levels. Separately, in solubilizing phosphatidyl-choline, either peptides with His residue at N-terminal position 2 or 3, or those containing Cys residue at position 11 or 12 were equipotent to peptide F3,6,14,1818A. Further, the lipid-solubilizing ability of those containing both His and Cys residues was comparable to that of peptide F3,6,14,1818A. In support of this, a similar structural importance was observed with Trp fluorescence study illustrating the penetration of peptides in phosphatidylcholine liposome. Besides, the modified peptides were also comparable to peptide F3,6,14,1818A in restoring phosphatidylserine-induced loss of PON1 activity. These results indicate that the insertion of His or Cys residue into peptide F3,6,14,1818A at appropriate positions could lead to enhanced antioxidant action with no significant change of lipid-solubilizing action.