Although additional work must be done at the molecular level to understand the precise signaling pathways induced upon P140 administration, these results show that in vivo P140 exerts a rapid effect on genes involved in inflammation and can restore some dysfunctions, as exemplified with PD-1. In conclusion, we have demonstrated that in MRL/lpr mice P140 binds to HSC70 and induces a novel regulatory circuit involving cd T cells, which affects autoreactive T and B cell survival. We thus propose a comprehensive mechanism triggered by P140 to control the lupus disease in this mouse strain. In addition, these findings shape our understanding of the immuno- regulatory functions of cd T cells in autoimmune diseases. Materials and Methods Synthetic peptides The synthesis and purification of P140 peptide (RIHM- VYSKRpSGKPRGYAFIEY; produced under Good Manufactur- ing Practice conditions), and of peptides 131–151 (RIHM- VYSKRSGKPRGYAFIEY) and ScP140 (YVSRYFGpSAIR- HEPKMKIYRG) were described previously [1,3,7]. For the biotin-labeled peptides, the biotin moiety was introduced at the end of peptide assembly as a Fmoc-Lys(biotin)-OH derivative. Homogeneity of peptides was checked by analytical high- performance liquid chromatography, and their identity was assessed by matrix-assisted laser desorption and ionization time- of-flight (TOF) mass spectrometry using a Protein TOF apparatus (Bruker Spectrospin). The solubility limits of both peptides in the cell culture medium RPMI 1640, PBS and distilled water were determined at 20 and 37uC using dynamic light scattering measurements [18]. 1H-NMR experiments D2O (99.8%) and trimethylsilyl-3-propionic acid-2,2,3,3-d4 (TSP-d4) were purchased from Euriso-top. Peptides 131–151 and P140 (2 mM final) were dissolved in 0.6 mL of 95% H2O and 5% D2O. The pH of the NMR sample was adjusted to 4.0. Spectra were recorded at 4uC using a Bruker DRX600 NMR spectrom- eter. All chemical shifts were referenced to methyl signals of TSP- d4 as an internal standard. 2D TOCSY experiments [41,42] were performed on each peptide with a mixing time, tm, of 20 and 70 ms. Four 2D-NOESY experiments with mixing times ranging from 100 to 500 ms were performed. The build-up curve [43] for different NOE correlations showed that spin diffusion was negligible for tm = 200 ms. The spectral width in F1 was 7200 Hz. Water resonance suppression in the TOCSY and NOESY experiments was achieved using the water suppression by gradient-tailored excitation (WATERGATE) sequence [44,45]. Data processing was performed using XWIN-NMR software. Distance restraints for structural calculations were generated from the NOESY spectra (tm = 200 ms) using XEASY software [46] and by classifying NOE cross-peaks as strong (1.8–2.5 A˚), medium (2.5–3.5 A˚) and weak (3.5–5.5 A˚) based on the volume of the respective cross-peaks [47], calculated by taking as reference the distance of 1.78 A˚ between two non-equivalent geminal protons. Molecular dynamics calculation Energy minimization (EM) and molecular dynamics (MD) calculations were performed on an Octane Silicon Graphics workstation. The structure calculations were done by restrained molecular dynamics in dihedral angle space using the standard simulated annealing protocol, as implemented in the program DYANA-1.4 [48] to generate, in a random way, a set of 100 initial structures corresponding to the NMR constraints. The SA P140 Peptide in Lupus protocol permits the exploration of the whole conformational space to find the global minimum energy conformation of the molecule and it has been shown to be a useful procedure for the study of constrained systems [49–51]. Fifty structures with the lowest energies were chosen for the refinement step with restrained minimization. Each of them was submitted to 900 steps of minimization (300 steps with steepest descent, 300 steps with conjugate gradient and 300 steps with Newton-Raphson methods), then to 35ps of MD in vacuo at 300K for equilibration and finally to 200ps of MD applying derived NMR restraints with a force constant of 20 kcal mol21 A˚22. Conjugate gradient EM was completed within 750 steps. The 50 refined structures were evaluated with MOLMOL [52] as results. For MD and EM calculations, the DISCOVER-3 module of the Insight II program (Accelrys, Inc.) was used. Consistent-valence force field with a default cut-off distance of 9.5 A˚ and a distance-dependent dielectric constant, equal to 4r, was applied to all peptides [53]. In order to avoid structural artefacts due to overestimation of coulombic interactions in vacuo, the net electric charges of the N- and C-terminal charged groups were neglected. The w angle for the non-glycine L-residues was constrained between 2175u and 0u. Identification of P140 receptor The detection of cell surface protein(s) recognized by P140 was adapted from a previously described method [54,55]. Total splenocytes and LN cells from 7–8 week-old MRL/lpr or unprimed CBA/J mice were isolated on nylon membrane (100 mM). Cells (about 506106) were incubated for 1h at room temperature (RT) in 5 mL of serum free RPMI 1640 culture medium (Biomedia) containing biotin-labeled peptides (P140 peptide, peptide 131–151 or scramble P140 peptide). In some experiments, unlabeled P140 peptide was added as a competitor. After washing cells once in phosphate buffered-saline (PBS) containing 1 mM EDTA, nucleus-free cell extracts were prepared in lysis buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mL/1mL protease inhibitors from Sigma, 0.5% Triton X100, 1 mM NaF, 1 mM Na3VO4). The complexes formed between the biotin-labeled P140 and putative cell-surface receptors were isolated by purification of the extracts using 100 mL avidin-agarose (ImmunoPure Immobilized Avidin, Pierce) in PBS containing 1 mM EDTA, 1mM NaF, and 1 mM Na3VO4. After overnight incubation at 4uC, the samples were washed extensively with the same buffer. The purified proteins were subjected to SDS-PAGE and revealed by colloidal blue staining. The relative signal intensity of each band was quantified by densitometry with the ImageJ 1.33u freeware (Gel Analyzer tool, NIH) after scanning. The identification of HSC70 was deduced from mass spectrometry analysis as described below after cutting bands of interest. Protein preparation for in-gel digestion The gel pieces were successively washed with 50 mL of 25 mM NH4HCO3 and 50 mL of acetonitrile (three times), and dehydrat- ed with 100 mL acetonitrile before reduction in the presence of 10 mM dithiothreitol in 25 mM NH4HCO3 (1h at 57uC) and alkylation in the presence of 55 mM iodoacetamide in 25 mM NH4HCO3. For tryptic digestion, the gel pieces were resuspended in 2 volumes of trypsin (12.5 ng/mL; Promega V5111) freshly diluted in 25 mM NH4HCO3 and incubated overnight at 37uC. The digested peptides were then extracted from the gel in a solution containing 34.9% H2O, 65% acetonitrile and 0.1% HCOOH, and directly analyzed by nanoLC-MS/MS. PLoS ONE | www.plosone.org 9 April 2009 | Volume 4 | Issue 4 | e5273