Structural statistics for the ensemble of the best 20 structures of human obestatin (1), its fragments and mouse obestatin (6).

<p>[a] The final CYANA target function value was computed for the structures calculated using CYANA.</p><p>[b] Average values of the 20 final energy-minimized CYANA conformers.</p><p>[c] Calculated using PROCHECK-NMR.</p><p>[d] Atomic differences are given as the average RMS difference of the mean coordinate structure (mean).</p

Analysis of the expression and functionality of GPR39 in ARPE-19 cells.

<p>(A) Immunocytochemical detection of GPR39 in ARPE-19 cells (objective magnification of 20x). (B) The effect of siRNA depletion of GPR39 on pAkt(S473) and pERK1/2(T202/Y204) in ARPE-19 cells after human obestatin treatment (<b>1</b>, 100 nM, 10 min). The ARPE-19 cells were transfected with GPR39 siRNA prior to obestatin <b>1</b> treatment. Equal amounts of protein in each sample were used to assess the expression of GPR39 by western blotting. The GPR39 level was expressed as the fold change relative to the control siRNA-transfected cells (mean ± SE). The protein expression was normalized relative to actin. The data are expressed as the mean ± SE. The asterisk (*) denotes <i>P</i><0.05 when comparing the treated control siRNA group with the control siRNA group; the dagger (#) denotes <i>P</i><0.05 when comparing the GPR39 siRNA group with the control siRNA group.</p

Primary structure of the obestatins used in this work: human obestatin (1), human non-amidated obestatin (2), human (6–23)-obestatin (3), human (11–23)-obestatin (4), human (16–23)-obestatin (5) and mouse obestatin (6).

<p>The red-labeled residues in <b>6</b> are different from those of <b>1</b>.</p

Immunocytochemical analysis of the Ki67 expression and BrdU incorporation in ARPE-19 cells.

<p>Immunocytochemical analysis of the Ki67 expression in ARPE-19 cells after 24 h of proliferation. A) Control. B) 10% FBS (v/v). C) 100 nM human obestatin (<b>1</b>). D) 100 nM human non-amidated obestatin (<b>2</b>). E) 100 nM human (6–23)-obestatin (<b>3</b>). F) 100 nM human (11–23)-obestatin (<b>4</b>). G) 100 nM human (16–23)-obestatin (<b>5</b>). H) 100 nM mouse obestatin (<b>6</b>). The magnification was 20x. I) Quantification of the immunocytochemical expression of Ki67 in ARPE-19 cells after treatment with 10% FBS (v/v; 100±2), 100 nM human obestatin (<b>1</b>; 84±1%), 100 nM human non-amidated obestatin (<b>2</b>; 33±2%), 100 nM human (6–23)-obestatin (<b>3</b>; 56±3%), 100 nM human (11–23)-obestatin (<b>4</b>; 67±3%), 100 nM human (16–23)-obestatin (<b>5</b>; 50±2%) and 100 nM mouse obestatin (<b>6</b>; 32±2%). The expression of Ki67 was expressed as the fold change relative to the expression level in FBS-treated cells in the positive control (mean ± SE). J) BrdU incorporation in ARPE-19 cells after treatment with 10% FBS (v/v; 100±1), 100 nM human obestatin (<b>1</b>; 84±4%), 100 nM human non-amidated obestatin (<b>2</b>; 39±2%), 100 nM human (6–23)-obestatin (<b>3</b>; 30±1%), 100 nM human (11–23)-obestatin (<b>4</b>; 52±3%), 100 nM human (16–23)-obestatin (<b>5</b>; 34±3%) and 100 nM mouse obestatin (<b>6</b>; 35±1%). The BrdU incorporation was expressed as the fold change relative to the level in FBS-treated cells in the positive control (mean ± SE). The data are expressed as the mean ± SE. The asterisk (*) denotes <i>P</i><0.05 when comparing the peptide-treated ARPE-19 cells groups with the human obestatin (<b>1</b>)-treated group.</p

Comparison between the secondary helical structure based on the H^N and H^alpha chemical shifts indices determined using the RCI server and the secondary helical structure obtained in our structures.

<p>The underlined residues were predicted to have secondary helical structure based on the H^N and H^alpha chemical shifts indices obtained using the RCI server (<a href="http://wishart.biology.ualberta.ca/rci" target="_blank">http://wishart.biology.ualberta.ca/rci</a>). The residues within helical structures that extended over more than two residues are represented in bold; these residues are located primarily at the C-terminus. The secondary helical structure obtained in our structures, as calculated by CYANA, included α-helix formation (green labels) and 3₁₀-helix formation (blue labels).</p

Summary of sequential and medium-range NOE connectivities involving the NH, Hα and Hβ protons of the peptides in SDS micelles, as derived from CYANA calculation.

<p>The thickness of the bar indicates the intensities of the NOEs for the following peptides: (A) human obestatin (<b>1</b>), (B) human non-amidated obestatin (<b>2</b>), (C) human (6–23)-obestatin (<b>3</b>), (D) human (11–23)-obestatin (<b>4</b>), (E) human (16–23)-obestatin (<b>5</b>) and (F) mouse obestatin (<b>6</b>). The asterisk (*) represents the C-terminal amidation of the molecule. The dagger (#) represents the differences between human obestatin (<b>1</b>) and mouse obestatin (<b>6</b>).</p

Secondary helical structure based on the H^N and H^alpha chemical shifts indices determined using the RCI server.

<p>The underlined residues were predicted to have secondary helical structure based on the H^N and H^alpha chemical shifts indices that were determined using the RCI server (<a href="http://wishart.biology.ualberta.ca/rci" target="_blank">http://wishart.biology.ualberta.ca/rci</a>). The residues within helical structures that extended over more than two residues are represented in bold; these residues are located primarily at the C-terminus.</p

Plot of the chemical shifts indices (CSIs) for residues at the C-termini of the studied peptides.

<p>(A) CSIs of the backbone amide protons (H^N) and (B) CSIs of the alpha protons (H^alpha). The peptide sequence (<b>6</b>) is aligned with that of the other peptides. The CSI is defined as δ^obs – δ^random-coil.</p

Secondary structure analyses performed using the DICHROWEB web server with the CONTINLL algorithm and reference data set 4.

<p>The relative amounts of α-helix and β-sheet were determined by adding together the contributions from helix 1 plus helix 2 and strand 1 plus strand 2, respectively, whereas the amounts of β-turn and random structure were read directly from the output. The peptides clearly showed greater helicity in SDS than in PBS.</p