Sequence alignment of human kallikreins (panel A) and three-dimensional structure of PSA (panel B).

<p>Sequence alignment (panel A) is built with those human kallikreins for which the three-dimensional structure is available at the Protein Data Bank. The protein sequences were obtained from the NCBI database (<a href="http://www.ncbi.nlm-nih.gov" target="_blank">http://www.ncbi.nlm-nih.gov</a>). The progressive multiple alignment of PSA (also named kallikrein 3; NCBI entry number: CAD30845.1), kallikrein 1 (also named tissue kallikrein; KLK1; NCBI entry number: AAH05313.1), kallikrein 2 (KLK2; NCBI entry number: AAF08276.1), kallikrein 4 (KLK4; NCBI entry number: AAD38019.1), kallikrein 6 (KLK6; NCBI entry number: AAP35498.1), kallikrein 7 (KLK7; NCBI entry number: NP_644806.1), and human plasma kallikrein (HPK; NCBI entry number: AAF79940.1) was performed by the Clustal-Omega program (<a href="http://www.ebi.ac.uk/Tools/msa/clustalo" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalo</a>). Only the trypsin-like serine protease domain of HPK has been aligned. The “*” symbol means that the residues are identical in all the aligned sequences; the ":" symbol indicate conserved substitutions, and the "." symbol means semi-conserved substitutions. The amino acid sequence of bovine chymotrypsinogen (BCTRP; NCBI entry number: 681083A) has been reported as the template. Three-dimensional structure of PSA (panel B). In both panels, the image was produced using UCSF Chimera molecular graphics package <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Pettersen1" target="_blank">[26]</a>. The “kallikrein loop” is in yellow <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Menez1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Tang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470-Fernndez1" target="_blank">[28]</a>, amino acid residues forming the catalytic triad are in red, and amino acid residues affecting the pH dependence of the catalytic parameters are in cyan.</p

Proton-linked equilibria for the enzymatic activity of PSA at 37°C.

<p>Proton-linked equilibria for the enzymatic activity of PSA at 37°C.</p

<i>p</i>K_a values from the pH-dependence of various kinetic parameters.

<p><i>p</i>K_a values from the pH-dependence of various kinetic parameters.</p

Time course of the PSA-catalyzed hydrolysis of Mu-HSSKLQ-AMC.

<p>Observation wavelength  = 460 nm, pH = 7.5 and temperature  = 37.0°C. The concentration of PSA was 50 nM. The concentration of Mu-HSSKLQ-AMC was 5 µM.</p

sLOX-1 release in EA.hy926 cells.

<p>(A) EA.hy926 cells were grown overnight in serum free medium in the absence (lanes 1–4) or in the presence (lanes 5–8) of ox-LDL (20μg/ml). MβCD was added to cells for the last 30 min incubation (lane 2, 3 and 8). GM6001 inhibitor was present for 30 min (lane 3) or overnight (lane 6). Western blot analysis of conditioned media was performed with anti-human LOX-1 receptor. (B) MTS assay performed on EA.hy926 cells plated at a density of 1,5x10⁴ cells/well and incubated overnight with different concentration of ox-LDL. Results are expressed as percentage of absorbance ± SEM compared to control cells.</p

Dependence of <i>k</i> (panel A) and <i>v</i> (panel B) on the substrate concentration for the PSA-catalyzed hydrolysis of Mu-HSSKLQ-AMC.

<p>The continuous lines fitting the data reported in panels A and B were obtained according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470.e003" target="_blank">Eqns. 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone.0102470.e004" target="_blank">4</a>, respectively, with values of <i>k</i>₂, <i>k</i>₃, and <i>K</i>_s (panel A), and of <i>k</i>_cat and <i>K</i>_m (panel B) reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102470#pone-0102470-t001" target="_blank">Table 1</a>. Values of pre-steady-state and steady-state parameters were obtained at pH 6.5 (o), pH 7.0 (x), pH 7.5 (+), pH 8.0 (*), pH 8.5 (∶), and pH 9.0 (⊕) at a temperature of 37.0°C.</p

Different parameters at various pH values, as obtained from the analysis of steady-state kinetics according to Eq. (1c) and of pre-steady-state kinetics according to Eq. (1d).

<p>Different parameters at various pH values, as obtained from the analysis of steady-state kinetics according to Eq. (1c) and of pre-steady-state kinetics according to Eq. (1d).</p

Immunoblotting of EA cells conditioned media probed with anti MMP-1 polyclonal mouse antibodies (upper panel), and TIMP-1 polyclonal mouse antibodies (lower panel).

<p>Optical density analysis was used to semi-quantify the protein secretion levels upon ox-LDL cell-treatment.</p

Competitive inhibitory effect of diazepam, diflunisal, ibuprofen, 3-indoxyl-sulfate, and propofol on the HSA-Tyr411-catalyzed hydrolysis of NphOHe (squares), NphODe (circles), and NphOMy (triangles).

<p>Data were obtained under conditions where [NphOHe] > 5×[HSA]. The analysis of data according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120603#pone.0120603.e010" target="_blank">equation (10)</a> allowed to determine values of <i>K</i>_I (indicated by arrows) reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120603#pone.0120603.g005" target="_blank">Fig. 5</a>. Where not shown, the standard deviation is smaller than the symbol. For details, see text.</p

sLOX-1 release in COS cells.

<p>(A) LOX-1-V5-COS transfected cells were treated with filipin (5μM) and glimepiride (5μM) for 15, 30 and 60 min. Conditioned media were centrifuged at 100,000 g and the resulting pellets (P100, lanes 2–4) and supernatants (S100, lanes 5–13) were analyzed by Western blotting. Lane 1 (Extr.) shows total protein extract (5 μg) loaded as a positive internal control of electrophoretic mobility of full-length LOX-1 receptor. (B) Upper panel shows sLOX-1 amount released in cells treated with Filipin (5μM) and MβCD (5mM) for 30 min compared to sLOX-1 constitutively released from untreated cells at 1, 2 and 5 hours. Lower panel shows the densitometric analysis for sLOX-1 band intensity. Data in histograms represent the average ± SEM of three experiments, P < 0.05 (*), P < 0.01 (**) or P < 0.001 (***).</p