Selectivity pocket found in hSIRT2.

<p><b>(A)</b> Superposition of LiSIR2rp1(ΔP253-E303)/Acetylated p53 peptide complex (grey) with hSIRT2 in complex with ADPR alone (pdb: 5d7o)(orange) or with EX243 inhibitor (pdb: 5d7p) (pink)(rmsd = 1.2Å). Upon binding of EX243, hSIRT2 active site rearranges and a new selectivity pocket forms. The hinge loop (residues 136–144) shifts of about 6Å. <b>(B)</b> and <b>(C)</b> Closer views of the hinge region of the structures shown in <b>(A)</b>. Residues of the hinge loop are depicted as sticks (first and second number refers to hSIRT2 and LiSIR2rp1, respectively).</p

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Multiple sequence alignments of LiSIR2RP1 with yeast Hst2 and human SIRT1-3 homologues.

<p>Multiple sequence alignment of LiSIR2rp1 (Uniprot #Q8I6E4) with ScHst2 (P53686), hSIRT1 (Q96EB6), hSIRT2 (Q8IXJ6) and hSIRT3 (Q9NTG7) (ScHst2 region 1–228 and 517–747 and hSIRT3 1–108 were omitted for clarity). Above the alignment, boxes define the Rossmann-fold (pink), and the small domain (blue). The main secondary structure elements described in ScHst2 (pdb: 1q14) and hSIRT2(pdb: 3zgo) are indicated. Important residues are pointed: residues important for binding of the NAD (blue circle), acetyl-lysine peptide (green circle) and Zinc ion (blue triangle) and identified as hot-spot by Parenti <i>et al</i>. (red circle) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193602#pone.0193602.ref056" target="_blank">56</a>]. ECS site and selectivity binding pocket are delimited by a red line. Results of globular domain predictions by GlobPlot and IUPred are depicted as orange boxes (full colored in case of consensus between both algorithm and hachured when identified by only one). Results of secondary structure predictions by NPS@ and PSIPRED are represented beneath (β sheets are indicated with arrows and α helices with cylinders).</p

LiSIR2rp1(ΔP253-E303)/Acetylated p53 peptide complex structure.

<p><b>(A)</b> Structure of LiSIR2rp1(ΔP253-E303) in complex with p53 peptide substrate. Large Rossmann-fold domain is colored in light pink, small domain in turquoise, cofactor binding loop in light orange and p53 peptide is depicted as sticks in red. <b>(B)</b> Electron densities (2Fo-Fc map contoured at 1σ) observed for acetylated p53 peptides (chains C and D) in both complexes of the asymmetric unit. Peptides are oriented as in D. <b>(C)</b> Surface representation of the LiSIR2rp1 in complex with p53 peptide (represented as sticks) binding to the cleft between the small and large domains. <b>(D)</b> Closer view of the interactions between LiSIR2rp1 and p53 peptide showing the three-stranded LiSIR2rp1-p53 β-sheet termed the β-staple (central substrate peptide strand surrounded on each side by β-strands contributed by both LiSIR2rp1 domains–Large Rossmann-fold and small zinc-binding domains) <b>(E)</b> Schematic representation of the interactions between LiSIR2rp1 and p53 peptide (residues numbered in red). Residues (single letter symbol with number) involved in main interactions through main chain (full line boxes) or side chain (dash line boxes) are represented. The red dashed lines indicate hydrogen bonds. Brown curve represents hydrophobic tunnel in which acetylated lysine inserts with surrounding residues implicated in hydrophobic and or van der Waals interactions with the acetyl-lysine. Residues belonging to the large Rossmann-fold domain and to the small domain are in pink and blue boxes, respectively.</p

Probing the proteolytically-stable domain structure of LiSIR2rp1 and its deacetylase activity.

<p><b>(A)</b> Primary amino acid sequence of 6 x His N-terminal tagged LiSIR2rp1 showing positions sensitive to tryptic digestion under non-denaturing conditions (arrows), defined from SDS PAGE and LC/ESI-TOF MS (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193602#sec002" target="_blank">Methods</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193602#pone.0193602.s009" target="_blank">S1 Text</a>). The larger arrows indicate principal digestion sites in time-resolved proteolysis. The proteolytically-sensitive region linking proteolytically-stable N-terminal (yellow) and C-terminal (blue) regions is shown in red. <b>(B)</b> Time-resolved tryptic digestion (0–20 min) of LiSIR2rp1 showing appearance of an N-terminal, ~29 kDa region (28580.83 and 28921.53 Da) and a ~ 9 kDa C-terminal region (9593.98, 9081.4, 7999.25 and 7710.43 Da) derived from an initial ~ 11 kDa digestion product (t = 1 to 3 min). (<i>The larger protein at 66 kDa is albumin in the quenching buffer</i>). <b>(C)</b> Re-purification of His tagged LiSIR2rp1 after native tryptic digestion; a 6 x His LiSIR2rp1 ΔN^5-373 mutant protein lacking the N-terminal trypsin site was used for these experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193602#sec002" target="_blank">Methods</a>). <b>Lanes 1</b> and <b>5</b>, undigested LiSIR2rp1 purified on Ni²⁺- affinity column. <b>Lane 2</b>, native digest of LiSIR2rp1 re-purified on Ni²⁺-affinity column (N- and C- termini bind). <b>Lane 3</b>, 6 M urea wash of native digest of LiSIR2rp1 bound to Ni²⁺- affinity column (C-terminal domain removed). <b>Lane 4</b>, Elution of native digest from Ni²⁺-affinity column with imidazole after 6 M urea wash (N-terminus alone). <b>(D)</b> Sirtuin deacetylase activity of LiSIR2rp1 after native tryptic digestion. Time resolved digests of 6 x His LiSIR2rp1 ΔN^5-373 were re-purified from trypsin reactions by Ni²⁺- affinity chromatography before assessing specific activity on the p53 acetyl lysine substrate. Specific activity (nmol deacetylated product/sec/mg protein) was compared for (i) undigested protein (t = 0) measured both for 6xhis LiSIR2rp1 ΔN^5-373 and full length protein (shown in parentheses), (ii) a 2.5 min digest and (iii) a 20 min digest. The fragmentation patterns were deduced from the results of SDS PAGE and LC/MS analyses.</p

Expression of LiSIR2rp1 truncated forms in promastigotes.

<p><b>(A)</b> Coomassie blue-stained SDS-PAGE gel of 5 μg of recombinant LiSIR2rp1 protein (WT) and respective truncated forms (ΔP253-E303; ΔP253-H322; ΔS272-S310; ΔS272-H322). <b>(B)</b> Western blot showing recognition of all LiSIR2rp1 recombinant forms by anti-L. major SIR2rp1 (α-LmSIR2rp1) IIIG4 monoclonal antibody (mAb). <b>(C)</b> Western blot analysis of LiSIR2rp1 levels in LiSIR2rp1 single knockout (sKO) promastigotes complemented with each pSPαBLASTα<i>Li</i>SIR2rp1 construct, as well as in sKO and WT parasites. Cysteine synthase (CS) was used as the loading control. <b>(D)</b> Quantification of pSPαBLASTα<i>Li</i>SIR2rp1 constructs by qPCR using genomic DNA of transfected, sKO and WT parasites, relative to sKO pSPαBLASTα<i>Li</i>SIR2rp1^WT. <b>(E, F)</b> Expression levels of LiSIR2rp1 <b>(E)</b> and the blasticidin resistance marker <b>(F)</b> determined by qPCR using cDNA from transfected parasites, relative to sKO pSPαBLASTα<i>Li</i>SIR2rp1^WT. Real time quantitative PCR (qPCR) analysis was performed using <i>LirRNA45</i> as the reference gene. Means and standard deviations of two independent experiments are represented. Unpaired t-test with Welch’s correction was performed: ***, p < 0.01; ****, p < 0.0001; n.s., non-significant.</p

NAD⁺-dependent deacetylase activity of recombinant LiSIR2rp1 protein with directed internal deletions within the β8-β9 connector.

<p>NAD⁺-dependent deacetylase activity of recombinant LiSIR2rp1 protein with directed internal deletions within the β8-β9 connector.</p

ROC analysis of normalised Act_A11 activity with PainOmics versus control serum samples.

<p>The Area Under the Curve (AUC) of 0.51 ± 0.06 indicates this marker is essentially invariant between serum controls versus PainOmics samples, thereby confirming the homogeneity of the various samples received.</p

Test of sample integrity.

<p> Normalised carboxypeptidase activity for PainOmics samples analysed (<i>A</i>) by centre and (<i>B</i>) as a whole PainOmics group versus control serum samples. (Control = 1.0 ± 0.1 versus PainOmics = 1.0 ± 0.2; unpaired t test <i>P</i> = 0.6). Each data point represents an individual patient sample (average of <i>n</i> = 8 replicates).</p

DNA concentration before and after validation.

<p>DNA concentrations (mean ± SD) of DNA test samples in duplicate. Dots indicate single DNA samples of Clinical Centres before and after SOP adjustment.</p