Screening of the binding properties of molecularly imprinted nanoparticles via capillary electrophoresis.

In response to the need for straightforward analytical methods to assess the affinity of molecularly imprinted nanoparticles (MIP NPs) for ligands, capillary electrophoresis (CE) was exploited using MIP NPs targeting the iron-regulating hormone hepcidin. In this work, MIP NPs were challenged with their template peptide, i.e., the N-terminal 5-mer of hepcidin, in comparison to unrelated ligand peptides. A CE separation method was developed ex novo achieving, after optimization of the background electrolyte (150 mM sodium phosphate pH 7.4) and of the running temperature (35 °C), the full separation of the free ligand from the complexed MIP NPs. The CE binding isotherm allowed the estimation of a micromolar dissociation constant for the 5-mer template-MIP NPs complex, in agreement with independent measurements. The CE offered the advantages of a direct injection of the MIP NPs/ligand incubation mix, without preliminary fractionation steps, requiring only minimal sample volumes and short analysis times. In conclusion CE proved to be a valid technique for characterizing the interactions of MIP NP libraries for selected target compounds.

Surface plasmon resonance based on molecularly imprinted nanoparticles for the picomolar detection of the iron regulating hormone Hepcidin-25.

BACKGROUND: Molecularly imprinted polymer (MIP) technique is a powerful mean to produce tailor made synthetic recognition sites. Here precipitation polymerization was exploited to produce a library of MIP nanoparticles (NPs) targeting the N terminus of the hormone Hepcidin-25, whose serum levels correlate with iron dis-metabolisms and doping. Biotinylated MIP NPs were immobilized to NeutrAvidin™ SPR sensor chip. The response of the MIP NP sensor to Hepcidin-25 was studied. FINDINGS: Morphological analysis showed MIP NPs of 20-50 nm; MIP NP exhibited high affinity and selectivity for the target analyte: low nanomolar Kds for the interaction NP/Hepcidin-25, but none for the NP/non regulative Hepcidin-20. The MIP NP were integrated as recognition element in SPR allowing the detection of Hepcidin-25 in 3 min. Linearity was observed with the logarithm of Hepcidin-25 concentration in the range 7.2-720 pM. LOD was 5 pM. The response for Hepcidin-20 was limited. Hepcidin-25 determination in real serum samples spiked with known analyte concentrations was also attempted. CONCLUSION: The integration of MIP NP to SPR allowed the determination of Hepcidin-25 at picomolar concentrations in short times outperforming the actual state of art. Optimization is still needed for real sample measurements in view of future clinical applications.

A Hot-Segment-Based Approach for the Design of Cross-Amyloid Interaction Surface Mimics as Inhibitors of Amyloid Self-Assembly.

The design of inhibitors of protein-protein interactions mediating amyloid self-assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self-assembly. Here we present a hot-segment-linking approach to design a series of mimics of the IAPP cross-amyloid interaction surface with Aß (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aß, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self- and cross-seeded IAPP self-assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer's disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self-assembly and pathogenic interactions of other proteins as well.

MIF-chemokine receptor interactions in atherogenesis are dependent on an N-loop-based 2-site binding mechanism.

Macrophage migration inhibitory factor (MIF) is a cytokine that mediates inflammatory diseases. MIF promotes atherogenic leukocyte recruitment through a promiscuous, yet highly affine, interaction with CXCR2 and CXCR4. Binding to CXCR2 is dependent on a pseudo-(E)LR motif in MIF, but a second interaction site has been elusive. Here we identified an N-like loop in MIF, suggesting that MIF binding to CXCR2 follows the 2-site binding mode of bona fide chemokines. For MIF, the model predicts interactions between the N-like loop and the CXCR2 N domain (site 1) and pseudo-(E)LR and extracellular loops (ELs) of CXCR2 (site 2). Applying biophysical and peptide array analysis, we demonstrated an interaction between MIF and the CXCR2 N domain, which was pseudo-(E)LR independent. Peptide array analysis also indicated that the pseudo-(E)LR motif is responsible for MIF binding to EL2 and 3. Notably, peptides MIF-(40-49) and MIF-(47-56), representing N-like-loop-derived peptides, but not a scrambled control peptide, significantly blocked MIF/CXCR2 binding, MIF-mediated monocyte arrest under flow on aortic endothelial cells in vitro (IC(50): 1.24×10(-6) M), and MIF-dependent monocyte adhesion to atherosclerotic mouse carotid arteries in vivo. Thus, the N-like loop in MIF is critical for MIF's noncognate interaction with CXCR2 and proatherogenic functions. The 2-site binding model that explains chemokine receptor activation also applies to MIF.

Dissecting the role of single regions of an IAPP mimic and IAPP in inhibition of Aß40 amyloid formation and cytotoxicity.

Alzheimer's disease (AD) and type 2 diabetes (T2D) are linked to the self-association of ß-amyloid peptide (Aß) and islet amyloid polypeptide (IAPP), respectively. We have shown that IAPP-GI, a soluble IAPP analogue and mimic of nonamyloidogenic and nontoxic IAPP, binds Aß with high affinity and blocks its cytotoxic self-assembly and fibrillogenesis. We have also shown that IAPP and Aß interact with each other into nonfibrillar and nontoxic heterocomplexes that suppress cytotoxic self-association by both polypeptides. The Aß-IAPP interaction might thus be a molecular link between AD and T2D. We studied the role of individual IAPP-GI and IAPP regions in their inhibitory function on Aß40 self-association and cytotoxicity. We found that the presence of the two hot-spot regions of the Aß-IAPP interaction interface in IAPP(8-28) is not sufficient for inhibitory function and that, in addition to IAPP(8-28), the presence of the N-terminal region IAPP(1-7) is absolutely required. By contrast, the C-terminal region, IAPP(30-37), is not required although its presence together with IAPP(1-7) in IAPP-GI results in a marked enhancement of the inhibitory effect as compared to IAPP(1-28)-GI. We suggest that the inhibitory effect of IAPP-GI and IAPP on Aß40 fibrillogenesis and cell toxicity is mediated primarily by interactions involving the hot regions of the Aß-IAPP interaction interface and the N terminus of IAPP while a concerted and likely structure-stabilizing action of the N- and C-terminal IAPP regions potentiates this effect. These results identify important molecular determinants of the amyloid suppressing function of the Aß40-IAPP interaction and could contribute to the design of novel inhibitors of Aß40 aggregation and cell degeneration.

Blocking Inflammasome Activation Caused by ß-Amyloid Peptide (Aß) and Islet Amyloid Polypeptide (IAPP) through an IAPP Mimic.

Inflammation in the brain and pancreas is linked to cell degeneration and pathogenesis of both Alzheimer's disease (AD) and type 2 diabetes (T2D). Inflammatory cascades in both tissues are triggered by the uptake of ß-amyloid peptide (Aß) or islet amyloid polypeptide (IAPP) aggregates by microglial cells (AD) or macrophages (T2D) and their insufficient lysosomal degradation. This results in lysosomal damage, caspase-1/NLRP3 inflammasome activation and release of interleukin-1ß (IL-1ß), a key proinflammatory cytokine in both diseases. Here we show that the inflammatory processes mediated by Aß and IAPP aggregates in microglial cells and macrophages are blocked by IAPP-GI, a nonamyloidogenic IAPP mimic, which forms high-affinity soluble and nonfibrillar hetero-oligomers with both polypeptides. In contrast to fibrillar Aß aggregates, nonfibrillar Aß/IAPP-GI or Aß/IAPP hetero-oligomers become rapidly internalized by microglial cells and targeted to lysosomes where Aß is fully degraded. Internalization occurs via IAPP receptor-mediated endocytosis. Moreover, in contrast to IAPP aggregates, IAPP/IAPP-GI hetero-oligomers become rapidly internalized and degraded in the lysosomal compartments of macrophages. Our findings uncover a previously unknown function for the IAPP/Aß cross-amyloid interaction and suggest that conversion of Aß or IAPP into lysosome-targeted and easily degradable hetero-oligomers by heteroassociation with IAPP mimics could become a promising approach to specifically prevent amyloid-mediated inflammation in AD, T2D, or both diseases.

Interaction between amyloid beta peptide and an aggregation blocker peptide mimicking islet amyloid polypeptide.

Assembly of amyloid-beta peptide (Aß) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimer's disease (AD) and interfering with Aß aggregation is an important strategy in the development of novel therapeutic approaches. Prior studies have shown that the double N-methylated analogue of islet amyloid polypeptide (IAPP) IAPP-GI, which is a conformationally constrained IAPP analogue mimicking a non-amyloidogenic IAPP conformation, is capable of blocking cytotoxic self-assembly of Aß. Here we investigate the interaction of IAPP-GI with Aß40 and Aß42 using NMR spectroscopy. The most pronounced NMR chemical shift changes were observed for residues 13-20, while residues 7-9, 15-16 as well as the C-terminal half of Aß--that is both regions of the Aß sequence that are converted into ß-strands in amyloid fibrils--were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aß resulted in a concentration-dependent co-aggregation of Aß and IAPP-GI that was enhanced for the more aggregation prone Aß42 peptide. On the basis of the reduced toxicity of the Aß peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aß into nontoxic "off-pathway" aggregates.

Crystal-state 3D-structural characterization of novel, Aib-based, turn and helical peptides.

The crystal-state conformations of the hexapeptide amide Pht-(Aib)(6)-NH-C(CH(3))(2)-O-OtBu (7), the hexapeptide Ac-L-aIle-(Aib)(5)-OtBu (6), the pentapeptide Z-(Aib)(3)-L-Glu(OtBu)-Aib-O-(CH(2))(2)-(1)Nap (5), the tetrapeptides Z-(Aib)(2)-L-His(N(tau)-Trt)-Aib-OMe (4 I) and Z-(Aib)(2)-L-Nva-Aib-OtBu (4 II), the tripeptide Pyr-(Aib)(3)-OtBu (3 I), the dipeptide amides Pyr-(Aib)(2)-(4)NH-TEMPO (3 II) and Piv-(Aib)(2)-NH-C(CH(3))(2)-O-OtBu (3 III), and the dipeptides Pht-Aib-betaAc(6)c-OtBu (2 I), Pht-Aib-NH-C(CH(3))(2)-O-OtBu (2 II) and Boc-gGly-mAib-OH (2 III) have been determined by X-ray diffraction analyses. All peptides investigated are characterized by one or more turn/helix forming Aib residues. Except the three short dipeptides, all are folded into C==O...H--N intramolecularly H-bonded 3(10)-helices, or into various types of beta-turns. In the structure of 6, two independent molecules of opposite screw sense were observed in the asymmetric unit, generating diastereomeric 3(10)-helices.

Handedness control of peptide helices by amino acid side-chain chirality: Ile/aIle peptides.

A set of four hexapeptide sequences, each characterized by four strongly helicogenic Aib residues and all combinations of two isomeric Ile/aIle residues at positions 2 and 5, was synthesized by solution methods and fully characterized. A detailed solution (by FT-IR absorption, NMR, and CD techniques) and solid/crystalline state (by X-ray diffraction) conformational investigation allowed us to validate our assumption that all four peptides are folded in well-developed 3(10)-helical structures. However, the most relevant conformational conclusion extracted from the present 3D-analysis is that the handedness of the 3(10)-helical structures formed does not seem to be sensitive to the configurational change at the beta-carbon atom of the constituent Ile versus the diastereomeric aIle residues (in other words, the dominant control on this important structural parameter appears to be exerted by the chirality of the amino acid alpha-carbon atom). These results complement published findings on the diverging relative stabilities of the intermolecularly H-bonded beta-sheet structures generated by Ile versus aIle homo-oligopeptides.