Erika Andreetto

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 (IC50: 1.24X10 “6 M), and MIF‐dependent monocyte adhesion to atherosclerotic mouse carotid arteries in vivo. Thus, the N‐like loop in MIF is critical for MIFs noncognate interaction with CXCR2 and proatherogenic functions. The 2‐site binding model that explains chemokine receptor activation also applies to MIF.—Kraemer, S., Lue, H., Zernecke, A., Kapurniotu, A., Andreetto, E., Frank, R., Lennartz, B., Weber, C., Bernhagen, J. MIF‐chemokine receptor interactions in atherogenesis are dependent on an N‐loop‐based 2‐site binding mechanism. FASEB J. 25, 894–906 (2011). www.fasebj.org

Interaction between Amyloid Beta Peptide and an Aggregation Blocker Peptide Mimicking Islet Amyloid Polypeptide

Assembly of amyloid-beta peptide (Aβ) into cytotoxic oligomeric and fibrillar aggregates is believed to be a major pathologic event in Alzheimers disease (AD) and interfering with Aβ 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 Aβ. Here we investigate the interaction of IAPP-GI with Aβ40 and Aβ42 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 Aβ - that is both regions of the Aβ sequence that are converted into β-strands in amyloid fibrils - were less accessible to solvent in the presence of IAPP-GI. At the same time, interaction of IAPP-GI with Aβ resulted in a concentration-dependent co-aggregation of Aβ and IAPP-GI that was enhanced for the more aggregation prone Aβ42 peptide. On the basis of the reduced toxicity of the Aβ peptide in the presence of IAPP-GI, our data are consistent with the suggestion that IAPP-GI redirects Aβ into nontoxic “off-pathway” aggregates.

Dissecting the Role of Single Regions of an IAPP Mimic and IAPP in Inhibition of Aβ40 Amyloid Formation and Cytotoxicity

Alzheimers disease (AD) and type 2 diabetes (T2D) are linked to the self‐association of β‐amyloid peptide (Aβ) and islet amyloid polypeptide (IAPP), respectively. We have shown that IAPP‐GI, a soluble IAPP analogue and mimic of nonamyloidogenic and nontoxic IAPP, binds Aβ with high affinity and blocks its cytotoxic self‐assembly and fibrillogenesis. We have also shown that IAPP and Aβ interact with each other into nonfibrillar and nontoxic heterocomplexes that suppress cytotoxic self‐association by both polypeptides. The Aβ–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 Aβ40 self‐association and cytotoxicity. We found that the presence of the two hot‐spot regions of the Aβ–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 Aβ40 fibrillogenesis and cell toxicity is mediated primarily by interactions involving the hot regions of the Aβ–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 Aβ40–IAPP interaction and could contribute to the design of novel inhibitors of Aβ40 aggregation and cell degeneration.

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 Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, 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 Alzheimers disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self-assembly and pathogenic interactions of other proteins as well.