Binding of the Hemopressin Peptide to the Cannabinoid CB₁ Receptor: Structural Insights

Hemopressin, a bioactive nonapeptide derived from the α1 chain of hemoglobin, was recently shown to possess selective antagonist activity at the cannabinoid CB1 receptor [Heimann, A. S., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 20588−20593]. CB1 receptor antagonists have been extensively studied for their possible therapeutic use in the treatment of obesity, drug abuse, and heroin addiction. In particular, many compounds acting as CB1 receptor antagonists have been synthesized and subjected to experiments as possible anti-obesity drugs, but their therapeutic application is still complicated by important side effects. Using circular dichroism and nuclear magnetic resonance spectroscopy, this work reports the conformational analysis of hemopressin and its truncated, biologically active fragment hemopressin(1−6). The binding modes of both hemopressin and hemopressin(1−6) are investigated by molecular docking calculations. Our conformational data indicate that regular turn structures in the central portion of hemopressin and hemopressin(1−6) are critical for an effective interaction with the receptor. The results of molecular docking calculations, indicating similarities and differences in comparison to the most accepted CB1 pharmacophore model, suggest the possibility of new chemical scaffolds for the design of new CB1 antagonist lead compounds

Aβ(25-35) aggregation on DOPC lipid bilayer.

<p>3×3 µm2 (a-c), and 5×5 µm2 (d-f), topographic AFM images (1024×1024px²) showing the time evolution of Aβ(25-35) on DOPC lipid bilayer. AFM measurements were performed in tapping mode and in PBS with a continuous scanning of the same area after the addition of the peptide. Over time, Aβ(25-35) forms layered aggregates (LA) on the bilayer surface, bilayer defect areas increase in size and Aβ(25-35) gradually covers the disrupted areas with disordered aggregates (DA). (g) Graph showing the increase in surface area covered by LA (blue line) and the evolution of lipid bilayer over time (red line). Data were qualitatively fitted by rational and sigmoidal functions for the DOPC bilayer (θDOPC) and the LA domains (θP), respectively. Fitting curves act as a guide-to-the-eye. (h) Height distribution histograms measured at t = 30 min (black), 1 h (red), 2 h (green), 3 h (blue), 4 h (cyan) and 5 h (magenta). The squared dashed-line in (e) indicates the area where statistical analyses on 5×5 µm2 AFM images were performed so as to keep the same scan size amongst images.</p

Aggregation of Aβ(25-35) on mica.

<p>Topographic AFM images of Aβ(25-35) aggregates grown on mica. Images were acquired in tapping mode and in PBS. (a) After 6 hours, Aβ(25-35) forms a smooth homogeneous layer without any well-formed aggregates (3×3 µm², 2459×2459px²). (b) On a smaller scale the peptide tends to organize into a texture with some discernible protrusions (white ellipse) (500×500 nm², 1228×1228px²).</p

Synthetic Peptides Reproducing Tissue Transglutaminase–Gliadin Complex Neo-epitopes as Probes for Antibody Detection in Celiac Disease Patients’ Sera

Celiac disease (CD) patients usually present high levels of circulating IgA antibodies directed to different antigens, in particular tissue transglutaminase (tTG), gliadin (Glia), and endomysium. A series of synthetic peptide constructs containing cross-linked tTG and Glia deamidated peptides have been synthesized. Peptides were tested in enzyme-linked immunosorbent assays against celiac disease patients’ sera versus normal blood donors, and their conformational features were evaluated by molecular modeling techniques. Four peptides were recognized as epitopes by autoantibodies (IgG class) circulating in CD patients’ sera before gluten-free diet. The peptide <b>II</b>, containing Ac-tTG(553–564)-NH₂ sequence cross-linked with deamidated Ac-α2-Glia(63–71)-NH₂, was able to identify specific disease antibodies with a sensitivity of 50% and a specificity of 94.4%. Structural conformations of the linear fragments Ac-tTG(553–564)-NH₂ and Ac-α2-Glia(63–71)-NH₂ and the corresponding cross-linked peptide <b>II</b> were calculated by molecular modeling. Results showed that cross-linking is determinant to assume conformations, which are not accessible to the linear fragments

Structures and Micelle Locations of the Nonlipidated and Lipidated C-Terminal Membrane Anchor of 2‘,3‘-Cyclic Nucleotide-3‘-phosphodiesterase^†

2,3‘-Cyclic nucleotide-3‘-phosphodiesterase (CNP) is a myelin-associated protein, an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2‘,3‘-cyclic nucleotides to produce 2‘-nucleotides, but the physiologically relevant in vivo substrate is still unknown. Recently, it was found that CNP is a possible linker protein between microtubules and the plasma membranes. Since CNP is modified post-translationally by an isoprenylation process at its C terminus, the prenylation is hypothesized to be a requisite process, which permanently anchors CNP to the plasma membrane. This study investigates the molecular mechanism of the interaction between CNP and the plasma membrane, proposing a general model to interpret the structural bases of prenylated proteins binding to the membrane. A 13 residue, C-terminal CNP fragment, C13, was demonstrated to be directly responsible for CNP membrane anchoring. C13 and its lipidated derivative (LIPO-C13) were subjected to conformational analysis in membrane mimetic environments, by means of CD and NMR spectroscopies. The orientation of C13 in relation to the membrane was investigated by NMR and EPR spin labeling studies. Our structural investigation shows that the presence of the lipidic tail is essential for the peptide to be folded and correctly positioned on the membrane surface. A general model is proposed in which the post-translational lipidation is an important biomolecular trick to enlarge the hydrophobic surface and to enable the contact of the protein with membrane

DOPC and DOPC/DHA lipid bilayers.

<p>(a) Height AFM images of DOPC (2.5×2.5 µm² 1024×1024px²) and DOPC/DHA (b) (2.5×2.5 512×512px²) lipid bilayers performed in tapping mode and in PBS. The bilayers cover 92% (DOPC) and 96% (DOPC/DHA) of the mica surface. On DOPC lipid particles collected at the border or within defects are due to incomplete bilayer formation or to defective rinsing.</p

Aβ(25-35) aggregation on DOPC/DHA lipid bilayer.

<p>(a-c) 5×2.2 µm² (1024×431px²) topographic AFM images of Aβ(25-35) aggregation on DOPC/DHA lipid bilayers. (d) Graph showing the increasing surface area covered by LA (blue line) and the lipid bilayer evolution over time (red line). LA structures were grown within the first 30 min of peptide deposition while the lipid surface area decreased slowly from 96% to 91%. Data were qualitatively fitted by rational and sigmoidal functions for the DOPC/DHA bilayer (Θ_DOPC/DHA) and the LA domains (Θ_P), respectively. Fitting curves act as guide-to-the-eye. (e) Height distribution histograms measured at t = 0 min (black), 30 min (red), 1 h (green), 1 h45 min (blue), 2 h (cyan) and 2 h20 min (magenta).</p

Aβ(25-35) high resolution analysis.

<p>Topographic AFM images (a, c) and corresponding phase images (b, d) performed in PBS on the same area (360×360 nm², 1024×1024 px²) of Aβ(25-35) LA domains on the DOPC bilayer. Images were acquired with an average tip-sample force of 170pN by scanning from left to right (a, b) and from right to left (c, d). Topographies clearly show both globular aggregates (circled) and annular structures (squared). The complex LA aggregate (arrow) in the bottom right-hand corner and the globular aggregate in the top right-hand corner (insets) are perturbed by the tip movement. (e, f) Height (black) and phase (red) line profiles of globular (e) and annular structures (f) measured along the white dashed line in Fig. 6a and 6c. (g,h) Height images of a highly dense globular structure region (286×286 nm², 574×574px²) acquired with two different tip-sample forces. At 220 pN (g), the globular structures are not perturbed, while at 234 pN (h) they are mechanically removed leaving the underlying annular structures. (i, l) AFM height images (770×770 nm², 633×633 px²) of LA on two different regions of the DOPC/DHA bilayer. After 1 h45 min of peptide deposition (i) the LA presents a linear organization highlighted by grey fibres. After 2 h20 min (l) the LA forms a structured layer where linear organization is less visible though still distinguishable. In some locations, it is organized into annular structures (red circles) similar in dimension but more sporadic and of different nature compared to the ones observed on DOPC.</p

Synthesis and Conformational Analysis of a Cyclic Peptide Obtained via <i>i</i> to <i>i</i>+4 Intramolecular Side-Chain to Side-Chain Azide−Alkyne 1,3-Dipolar Cycloaddition

Intramolecular side-chain to side-chain cyclization is an established approach to achieve stabilization of specific conformations and a recognized strategy to improve resistance toward proteolytic degradation. To this end, cyclizations, which are bioisosteric to the lactam-type side-chain to side-chain modification and do not require orthogonal protection schemes, are of great interest. Herein, we report the employment of CuI-catalyzed 1,3-dipolar cycloaddition of side chains modified with azido and alkynyl functions and explore alternative synthetic routes to efficiently generate 1,4-disubstituted  [1,2,3]triazolyl-containing cyclopeptides. The solid-phase assembly of the linear precursor including ϵ-azido norleucine and the propargylglycine (Pra) in positions i and i+4, respectively, was accomplished by either subjecting the resin-bound peptide to selective on-resin diazo transformation of a Lys into the Nle(ϵ-N3) or the incorporation of Fmoc-Nle(ϵ-N3)-OH during the stepwise build-up of the resin-bound peptide 1b. Solution-phase CuI-catalyzed 1,3-dipolar cycloaddition converts the linear precursor Ac-Lys-Gly-Nle(ϵ-N3)-Ser-Ile-Gln-Pra-Leu-Arg-NH2 (2) into the 1,4-disubstituted [1,2,3]triazolyl-containing cyclopeptide [Ac-Lys-Gly-Xaa(&1)-Ser-Ile-Gln-Yaa(&2)-Leu-Arg-NH2][(&1(CH2)4-1,4-[1,2,3]triazolyl-CH2&2)] (3). The conformational preferences of the model cyclopeptide 3 (III), which is derived from the sequence of a highly helical and potent i to i+4 side-chain to side-chain lactam-containing antagonist of parathyroid hormone-related peptide (PTHrP), are compared to the corresponding lactam analogue Ac[Lys13(&1),Asp17(&2)]hPTHrP(11−19)NH2 (II). CD and NMR studies of 3 and II in water/hexafluoroacetone (HFA) (50:50, v/v) revealed a high prevalence of turn-helical structures involving in particular the cyclic regions of the molecule. Despite a slight difference of the backbone arrangement, the side-chains of Ser, Gln, and Ile located at the i+1 to i+3 of the ring-forming sequences share the same spatial orientation. Both cyclopeptides differ regarding the location of the turn-helical segment, which in II involves noncyclized residues while in 3 it overlaps with residues involved in the cyclic structure. Therefore, the synthetic accessibility and conformational similarity of i to i+4 side-chain to side-chain cyclopeptide containing the 1,4-disubstituted [1,2,3]triazolyl moiety to the lactam-type one may result in similar bioactivities

Designed Glucopeptides Mimetics of Myelin Protein Epitopes As Synthetic Probes for the Detection of Autoantibodies, Biomarkers of Multiple Sclerosis

We previously reported that CSF114­(Glc) detects diagnostic autoantibodies in multiple sclerosis sera. We report herein a bioinformatic analysis of myelin proteins and CSF114­(Glc), which led to the identification of five sequences. These glucopeptides were synthesized and tested in enzymatic assays, showing a common minimal epitope. Starting from that, we designed an optimized sequence, SP077, showing a higher homology with both CSF114­(Glc) and the five sequences selected using the bioinformatic approach. SP077 was synthesized and tested on 50 multiple sclerosis patients’ sera, and was able to detect higher antibody titers as compared to CSF114­(Glc). Finally, the conformational properties of SP077 were studied by NMR spectroscopy and structure calculations. Thus, the immunological activity of SP077 in the recognition of specific autoantibodies in multiple sclerosis patients’ sera may be ascribed to both the optimized design of its epitopic region and the superior surface interacting properties of its C-terminal region