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Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I.

ABSTRACT: The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1-PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI ("target peptide") binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr(20) ? Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the target peptide (K(d) = 37.0 ?m), albeit with ?10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr(253) ? Ala substitution. Comparison of the 1.5-? resolution crystal structure of PDZ3 with its bound target peptide ((505)QEAKL(509)) to that of peptide-bound PDZ1 indicated fewer target peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr(20) ? Ala (PDZ1) + Tyr(253) ? Ala (PDZ3)) substitution abrogated all target peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr(253) ? Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr(20) ? Ala (PDZ1) + Tyr(253) ? Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.

SUBMITTER: Kocher O

PROVIDER: S-EPMC3137089 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I.

Kocher Olivier O Birrane Gabriel G Yesilaltay Ayce A Shechter Sharon S Pal Rinku R Daniels Kathleen K Krieger Monty M

The Journal of biological chemistry 20110523 28

The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1-PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI ("target peptide") binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr( ...[more]

PMID: 21602281

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Project description:BackgroundHigh-density lipoprotein (HDL) plays an antiatherogenic role by mediating reverse cholesterol transport (RCT), antioxidation, anti-inflammation, and endothelial cell protection. Recently, series of evidence have shown that HDL can also convert to proatherogenic HDL under certain circumstances. Plasma paraoxonase 1 (PON1) as an HDL-bound esterase, is responsible for most of the antioxidant properties of HDL. However, whether PON1 can serve as a therapeutic target of dysfunctional HDL-related atherosclerosis remains unclear.MethodsIn this study, scavenger receptor class B type I deficient (Scarb1-/- ) mice were used as the animal model with dysfunctional HDL and increased atherosclerotic susceptibility. Hepatic PON1 overexpression and secretion into circulation were achieved by lentivirus injection through the tail vein. We monitored plasma lipids levels and lipoprotein profiles in Scarb1-/- mice, and measured the levels and activities of proteins associated with HDL function. Meanwhile, lipid deposition in the liver and atherosclerotic lesions was quantified. Hepatic genes relevant to HDL metabolism and inflammation were analyzed.ResultsThe results showed the relative levels of PON1 in liver and plasma were increased by 1.1-fold and 1.6-fold, respectively, and mean plasma PON1 activity was increased by 63%. High-level PON1 increased the antioxidative and anti-inﬂammatory properties, promoted HDL maturation and macrophage cholesterol efflux through increasing HDL functional proteins components apolipoprotein A1 (APOA1), apolipoprotein E (APOE), and lecithin-cholesterol acyltransferase (LCAT), while decreased inflammatory protein markers, such as serum amyloid A (SAA), apolipoprotein A4 (APOA4) and alpha 1 antitrypsin (A1AT). Furthermore, hepatic PON1 overexpression linked the effects of antioxidation and anti-inflammation with HDL metabolism regulation mainly through up-regulating liver X receptor alpha (LXRα) and its downstream genes. The pleiotropic effects involved promoting HDL biogenesis by raising the level of APOA1, increasing cholesterol uptake by the liver through the APOE-low density lipoprotein receptor (LDLR) pathway, and increasing cholesterol excretion into the bile, thereby reducing hepatic steatosis and aorta atherosclerosis in Western diet-fed mice.ConclusionsOur study reveals that high-level PON1 improved dysfunctional HDL and alleviated the development of atherosclerosis in Scarb1-/- mice. It is suggested that PON1 represents a promising target of HDL-based therapeutic strategy for HDL-related atherosclerotic cardiovascular disease.

Dataset Information

Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I.

Publications

Identification of the PDZ3 domain of the adaptor protein PDZK1 as a second, physiologically functional binding site for the C terminus of the high density lipoprotein receptor scavenger receptor class B type I.

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