Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret

Abstract

Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases.

Keywords: CD26/DPPIV; Triuret; Uric acid.

Figure 1

Uric acid inhibition of cell bound CD26/DPPIV activity is competitive and requires cellular uptake of uric acid. Human recombinant CD26/DPPIV is not inhibited by uric acid, allantoin, or 6-aminouracil HUVEC were cultured, collected by trypsinization and then assayed for CD26/DPPIV activity. (A) HUVEC were incubated with the indicated concentration of uric acid and 60 µM Gly-Pro-AMC. Cleavage was recorded with a 360 nm excitation filter and a 460 nm emission filter. *P<0.0025 compared to 0 mg/dl uric acid. (n = 6. Error bars ± 1 s.d.) (B) HUVEC were incubated with the indicated concentration of Gly-Pro-AMC in the presence (grey bars) and absence (open bars) of 15 mg/dl uric acid. Each point is in triplicate shown ± standard deviation. *P < 0.05 compared to no uric acid. (C) 60 µM Gly-Pro-AMC was added to HUVEC in the presence or absence of 15 mg/dl of uric acid and/or probenecid (1 mM) as indicated. * P = 0.00003. (n = 5. Error bars ± 1 s.d.). (D) Uric acid (15 mg/dl) was added to human recombinant CD26/DPPIV (rCD26/DPPIV) (0.0625 ng/µl). All replicates for the 4 times the experiment has been repeated is shown (n = 9. Error bars ± 1 s.d.).

Figure 2

Triuret can inhibit recombinant CD26/DPPIV and uric acid’s inhibition of cell bound CD26/DPPIV is dependent on the intracellular formation of triuret. (A) rCD26/DPPIV was incubated with PBS in the absence (control) or presence of allantoin (0.892 mM) or in 2.67 mM KOH in PBS in the absence (KOH) or presence of 6-aminouracil (0.892 mM), as indicated, and 60 µM Gly-Pro-AMC. Each point is in triplicate shown ± standard deviation.(B) Triuret (2 mM) was added to human recombinant CD26/DPPIV. *P = 0.001 (n = 7. Error bars ± 1 s.d.). (C) Allantoin (0.892 mM) in PBS, 6-aminouracil (0.892 mM) in KOH, and triuret (2 mM) in PBS, KOH, or PBS were added to HUVEC *P = 0.028. (n = 3. Error bars ± 1 s.d.).

Figure 3

Uric acid’s ability to inhibit CD26/DPPIV activity is blocked with MnTBAP and enhanced with hypochlorous acid (A) HUVEC were treated with 40 µM MnTBAP for 24 hours prior to harvest. Collected cells were incubated in 60 µM Gly-Pro-AMC in the presence or absence of 15 mg/dl of uric acid as indicated.. All samples with uric acid added are shown as a percentage of pmoles of Gly-Pro-AMC cleaved/sec of control (no uric acid, same condition), normalized to 100%. * P = 0.009. (n = 5. Error bars ± 1 s.d.) (B) HUVEC were treated with 100 µM hypochlorous acid or control for 15 min, washed with PBS, and were kept in culture media at 37°C for 30 min prior to harvest. Cells were collected and analyzed as in (A). *P = 0.0026. (n = 5. Error bars ± 1 s.d.)

Figure 4

Modeling of triuret in the CD26/DPPIV active site and crystal structures of CD26/DPPIV complexed with some of its known inhibitors. (A) The crystal structures of small molecule inhibitors bound in the active site of CD26/DPPIV were used as references (PDB ID’s 2G5P, 2G5T, 2G63, 2I03). The central carbonyl oxygen of triuret was first structurally aligned with the common carbonyl oxygen of each of the inhibitors using the graphics program Coot {Emsley, 2004 #;Krissinel, 2004 #}. Steric interactions were then minimized by allowing for rotational freedom about the C-N bond of the terminal amide groups. (B) An overlay of crystal structures of four small molecule inhibitors of CD26/DPPIV (PDB ID 2G5P (magenta), 2G5T (blue), 2G63 cyan), and 2I03 (orange).