Effects of loss of PDZK1 on coronary atherosclerosis and cardiac fibrosis in apoE KO mice.

<p>Hearts were harvested from Paigen diet-fed mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008103#s2" target="_blank">Methods</a> (n = 8 per genotype). Left panels: A–B: representative cross-sections of Oil red O-stained (A–B) or trichrome-stained (C) myocardial coronary arterioles, showing unremarkable arterioles in apoE KO (A) and totally occluded arterioles in PDZK1/apoE dKO (B–C) mice (magnification, ×100). The Oil red O stain shows that the coronary arteriole is occluded almost exclusively by lipid-rich lesions (B), while the trichrome stain shows that the arteriole is surrounded by fibrosis in an area of myocardial infarction in a PDZK1/apoE dKO mouse (C). D–E: trichrome stained sections of hearts showing areas of infarction/fibrosis stained blue in PDZK1/apoE dKO (E), while they are absent in apoE KO (D) mice (magnification, ×10). Right top panel: quantification of coronary artery occlusions in apoE KO and PDZK1/apoE dKO mice. Statistically significant differences by ANOVA Tukey posthoc test comparing the two genotypes within a given group are indicated as: P<0.001. Right bottom panel: quantification of cardiac fibrosis in apoE KO and PDZK1/apoE dKO mice. Unpaired Student's t-test was used to determine statistical significance.</p

Lipoprotein cholesterol profiles from apoE KO and PDZK1/apoE dKO mice.

<p>Plasma harvested from individual mice fed a Paigen diet for three months was size fractionated using FPLC, and the total cholesterol contents of the fractions (mg/dL plasma) were determined by enzymatic assay. Profiles averaged from 2 independent experiments for each genotype, each composed of pooled plasma from six apoE KO (open circles) and six PDZK1/apoE dKO (filled circles) mice per experiment are shown. Approximate elution positions of human VLDL, IDL/LDL and HDL are indicated.</p

Aortic root atherosclerosis in Paigen diet-fed apoE KO and PDZK1/apoE dKO mice.

<p>Hearts were harvested from Paigen diet-fed apoE KO (A, C–D) and PDZK1/apoE dKO (B, E–F) mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008103#s2" target="_blank">Methods</a> (n = 8 per genotype). Left top panels: A–B: representative cross-sections of Oil red O-stained aortic root lesions. (magnification, ×20). Right top panel: quantification of aortic root atherosclerosis by planimetry. Unpaired Student's t-test was used to determine statistical significance. Bottom panels: immunohistochemistry of aortic root atherosclerotic plaques using CD68 (C and E) or alpha-smooth muscle actin α-SMA) (D and F) antibodies show that macrophages compose the overwhelming cell population of aortic root atherosclerotic plaques and that smooth muscle cells are rare in both apoE KO (C–D) and PDZK1/apoE dKO (E–F) mice. “L” indicates the vascular lumen, arrows indicate representative positive cells (magnification, ×100).</p

Plasma lipid levels and body weights of apoE KO and PDZK1/apoE dKO mice.

<p>Four month old animals were fasted for 4 hours prior to sample collection. Values are represented as mean±standard error from 8–25 animals (mean 20 animals) per group. Statistical significance was determined by pairwise comparisons of each value from PDZK1/apoE dKO mice with apoE KO controls by using unpaired Student's t test. The abbreviations and units used are TC (total plasma cholesterol, mg/dL), UC (plasma unesterified cholesterol, mg/dL), UC:TC (plasma unesterified cholesterol to total plasma cholesterol ratio), PL (plasma phospholipids, mg/dL), TG (plasma triglycerides, mg/dL) and Wt (body weights, g).</p>a, b, c, e<p>P<0.01</p>d<p>P<0.05</p

Immunoblot analysis of hepatic SR-BI expression.

<p>Mice with the indicated genotypes were fed a high fat/high cholesterol/cholate-containing “Paigen” diet for three months. Livers were harvested and subjected to immunoblotting using anti-SR-BI and anti-actin (loading control) antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008103#s2" target="_blank">Materials and Methods</a>.</p

Steps involved in the isoprenylation and processing of the Prostacyclin Receptor.

<p>The prostacyclin receptor (IP) contains an evolutionary conserved ‘<i>CaaX</i> motif’ at its cytoplasmic carboxy-terminus, <i>e.g</i> corresponding to C⁴¹⁴SLC⁴¹⁷ of the mouse IP as shown. During its processing, (i) the IP undergoes isoprenylation through <i>thio-ether</i> attachment of a carbon (C)-15 farnesyl moiety to Cys⁴¹⁴ while subsequent (ii) proteolytic cleavage, or <i>aaX</i>ing, liberates the terminal ⁴¹⁵<i>SLC</i>⁴¹⁷ residues and (iii) end-stage carboxy-methylation of the nascent α-carboxy-group on Cys⁴¹⁴ generates the fully processed, mature IP in its farnesyl-Cys-carboxymethylated form. Herein, the interaction of peptides based on the mouse IP carboxy-terminus with PDZ domain 1 (PDZ1) or full length PDZK1 was investigated through isothermal titration calorimetry (ITC) where Peptide 1 is a nanopeptide containing the seven carboxy-terminal amino acids (KK⁴¹¹IAACSLC⁴¹⁷); Peptide 2 is an octapeptide corresponding to the carboxy-terminus of IP (K⁴⁰⁷SEAIAAC⁴¹⁴) devoid of the 3 terminal amino acids (-<i>aaX</i>/-⁴¹⁵<i>SLC</i>⁴¹⁷) which are proteolytically cleavage following farnesylation of the IP; Peptide 3 is identical to Peptide 2 (K⁴⁰⁷SEAIAAC⁴¹⁴) except that it was modified by the addition of a C-15 farnesyl group on the carboxy-terminal cysteine (Cys⁴¹⁴) and a carboxy-methyl group on the terminal -α-COOH, thereby representing the farnesyl-Cys-carboxymethyl ester form of the C-terminus of the IP.</p

Structure determination and refinement statistics.

a<p>Values in parenthesis are for the highest resolution shell. R_sym = Σ|I-<i>|/Σ(I), where I is the observed integrated intensity, <i> is the average integrated intensity obtained from multiple measurements, and the summation is over all observable reflections. R_cryst = Σ||F_obs|-<i>k</i>|F_calc||/Σ|F_obs|, where F_obs and F_calc are the observed and calculates structure factors, respectively. R_free is calculated as R_cryst using 5% of the reflection chosen randomly and omitted from the refinement calculations. Bond lengths and angles are root-mean-square deviations from ideal values.</i></i></p

Isothermal titration calorimetric analysis of the binding of a C-terminal peptide from IP to the PDZ1 domain of PDZK1.

<p>Recombinant wild-type PDZ1 domain (0.03 mM in 150 mM NaCl, 0.5 mM tris (2-carboxyethyl) phosphine, 25 mM Tris-pH 8.0) were placed in the titration cell and equilibrated at 20°C. A solution containing 1.0 mM of (A) the C-terminal nonapeptide from IP, KK⁴¹¹IAACSLC⁴¹⁷, or (B) the octapeptide K⁴⁰⁷SEAIAAC⁴¹⁴ corresponding to the C-terminal sequence of IP from which the last three amino acid –SLC are absent, or (C) the farnesylated-carboxy-methylated form of the same peptide (K⁴⁰⁷SEAIAAC⁴¹⁴⁾ were injected in 10 µl aliquots with an interval of 4 minutes between each addition to permit re-equilibration. Titration curves were analyzed and K_d values determined using ORIGIN 7.0 software.</p

X-ray crystal structure of the PDZ1-IP target peptide chimera.

<p><b>A</b>: Amino acid sequence of the recombinant chimeric protein used for crystallization: N-terminal Gly-Ser dipeptide derived from the cloning vector (black), PDZ1 domain (residues 7–86 (PDZK1 numbering), green), partial interdomain segment (87–106, lies between the PDZ1 and PDZ2 domains, blue) and seven carboxy-terminal residues of IP (⁴¹¹IAACSLC⁴¹⁷, IP numbering, yellow). Regions of secondary structure (β strands and α helices) and the carboxylate-binding loop (CBL) are indicated above the sequence. <b>B</b>: Asymmetric unit showing the head-to-tail arrangement of two chimeric molecules. This figure was generated using POVScript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053819#pone.0053819-Fenn1" target="_blank">[47]</a> using the color scheme in panel A.</p

Structure of the C-terminal IP target peptide binding to PDZ1.

<p><b>A</b>: Ribbon diagram showing the three dimensional structure of PDZ1 (residues 7–86, green with gray carboxylate-binding loop) and the bound C-terminus of IP (⁻⁶IAACSLC⁰, yellow) from an adjacent molecule in the asymmetric unit. Six β-strands (β1-β6), two α-helices (α1–α2) and carboxylate-binding loop (dark gray) are indicated. Vector derived residues have been omitted for clarity. <b>B</b>: Two-dimensional representation of interactions between PDZ1 (green) and the C-terminal IP target peptide (yellow). Hydrogen bonds are shown as dashed lines and hydrophobic interactions as arcs with radial spokes. This figure was generated using LIGPLOT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053819#pone.0053819-Wallace1" target="_blank">[48]</a>. <b>C</b>: Stereo representation of the ligand-binding groove of PDZ1 (green) and the IP target peptide (yellow). Oxygen, nitrogen and waters molecules are shown in red, dark blue and cyan, respectively. Sulfur atoms are colored in yellow. Hydrogen bonds are shown as dashed lines. The orientation is similar to that in panel A.</p