The role of copper(II) and zinc(II) in the degradation of human and murine IAPP by insulin‐degrading enzyme

Amylin or islet amyloid polypeptide (IAPP) is a 37‐residue peptide hormone secreted from the pancreatic islets into the blood circulation and is cleared by peptidases in the kidney. IAPP aggregates are strongly associated with β‐cell degeneration in type 2 diabetes, as demonstrated by the fact that more than 95% of patients exhibit IAPP amyloid upon autopsy. Recently, it has been reported that metal ions such as copper(II) and zinc(II) are implicated in the aggregation of IAPP as well as able to modulate the proteolytic activity of IAPP degrading enzymes. For this reason, in this work, the role of the latter metal ions in the degradation of IAPP by insulin‐degrading enzyme (IDE) has been investigated by a chromatographic and mass spectrometric combined method. The latter experimental approach allowed not only to assess the overall metal ion inhibition of the human and murine IAPP degradation by IDE but also to have information on copper‐ and zinc‐induced changes in IAPP aggregation. In addition, IDE cleavage site preferences in the presence of metal ions are rationalized as metal ion‐induced changes in substrate accessibility. Copyright © 2014 John Wiley & Sons, Ltd.     

Peptide Nanotubes

The self‐assembly of different classes of peptide, including cyclic peptides, amyloid peptides and surfactant‐like peptides into nanotube structures is reviewed. The modes of self‐assembly are discussed. Additionally, applications in bionanotechnology and synthetic materials science are summarized.     

Interaction with the Surrounding Water Plays a Key Role in Determining the Aggregation Propensity of Proteins

Understanding the molecular determinants of the relative propensities of proteins to aggregate in a cellular environment is a central issue for treating protein‐aggregation diseases and developing peptide‐based therapeutics. Despite the expectation that protein aggregation can largely be attributed to direct protein–protein interactions, a crucial role the surrounding water in determining the aggregation propensity of proteins both in vitro and in vivo was identified. The overall protein hydrophobicity, defined solely by the hydration free energy of a protein in its monomeric state sampling its equilibrium structures, was shown to be the predominant determinant of protein aggregation propensity in aqueous solution. Striking discrimination of positively and negatively charged residues by the surrounding water was also found. This effect depends on the protein net charge and plays a crucial role in regulating the solubility of the protein. These results pave the way for the design of aggregation‐resistant proteins as biotherapeutics.     

Modulating Aβ33–42 Peptide Assembly by Graphene Oxide

Graphene oxide (GO) is utilized as the modulator to tune the formation and development of amyloid fibrils (Aβ_33–42). Atomic force microscopy temporal evolution measurements reveal that the initial binding between the peptide monomer and the large available surface of the GO sheets can redirect the assembly pathway of amyloid beta. The results support the possibility to develop graphene‐based materials to inhibit amyloidosis.     

Characterizing Methyl‐Bearing Side Chain Contacts and Dynamics Mediating Amyloid β Protofibril Interactions Using 13Cmethyl‐DEST and Lifetime Line Broadening

Many details pertaining to the formation and interactions of protein aggregates associated with neurodegenerative diseases are invisible to conventional biophysical techniques. We recently introduced ¹⁵N dark‐state exchange saturation transfer (DEST) and ¹⁵N lifetime line‐broadening to study solution backbone dynamics and position‐specific binding probabilities for amyloid β (Aβ) monomers in exchange with large (2–80 MDa) protofibrillar Aβ aggregates. Here we use ¹³C_methyl DEST and lifetime line‐broadening to probe the interactions and dynamics of methyl‐bearing side chains in the Aβ‐protofibril‐bound state. We show that all methyl groups of Aβ40 populate direct‐contact bound states with a very fast effective transverse relaxation rate, indicative of side‐chain‐mediated direct binding to the protofibril surface. The data are consistent with position‐specific enhancements of ¹³C_methyl‐${R{{{\rm tethered}\hfill \atop 2\hfill}}}$ values in tethered states, providing further insights into the structural ensemble of the protofibril‐bound state.     

Rational Design and Identification of a Non‐Peptidic Aggregation Inhibitor of Amyloid‐β Based on a Pharmacophore Motif Obtained from cyclo[‐Lys‐Leu‐Val‐Phe‐Phe‐]

Inhibition of pathogenic protein aggregation may be an important and straightforward therapeutic strategy for curing amyloid diseases. Small‐molecule aggregation inhibitors of Alzheimer’s amyloid‐β (Aβ) are extremely scarce, however, and are mainly restricted to dye‐ and polyphenol‐type compounds that lack drug‐likeness. Based on the structure‐activity relationship of cyclic Aβ16–20 (cyclo‐[KLVFF]), we identified unique pharmacophore motifs comprising side‐chains of Leu², Val³, Phe⁴, and Phe⁵ residues without involvement of the backbone amide bonds to inhibit Aβ aggregation. This finding allowed us to design non‐peptidic, small‐molecule aggregation inhibitors that possess potent activity. These molecules are the first successful non‐peptidic, small‐molecule aggregation inhibitors of amyloids based on rational molecular design.     

生物凝聚态物质的多层次结构表征

在生物体内到处都是由蛋白质、核酸和多糖等生物大分子构成的各种不同生物凝聚态物质,这些生物凝聚态物质形成不同的高级结构,执行不同的生物功能。获取这些生物凝聚态物质的高分辨结构是理解生命过程的重要途径。在离体环境中,获取高分辨结构的手段主要有X-射线晶体衍射、冷冻电镜和核磁共振等,而在活细胞内原位研究生物凝聚体的结构,核磁共振和化学交联质谱具有独特优势。本文总结了利用多种分析手段对生物凝聚态物质进行多层次结构表征的研究进展:包括简单纯化体系下的蛋白质分子机器,蛋白质纤维等;液-液相分离,大分子拥挤、限域等模拟细胞复杂环境下的生物大分子以及活细胞内生物大分子。     

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory‐Impairing Anti‐Parallel Aβ Dimers

Amyloid‐β (Aβ) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aβ oligomers. Herein parallel and anti‐parallel variants of Aβ(1–40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti‐parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA‐approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aβ oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aβ in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aβ interactions with dimer‐specific molecules.     

Interactions of human islet amyloid polypeptide with lipid structure of different curvatures

Curvature is one of the most important features of lipid membranes in living cells, which significantly influences the structure of lipid membranes and their interaction with proteins. Taken the human islet amyloid polypeptide (hIAPP), an important protein related to the pathogenesis of type II diabetes, as an example, we performed molecular dynamics (MD) simulations to study the interaction between the protein and the lipid structures with varied curvatures. We found that the lipids in the high curvature membrane pack loosely with high mobility. The hIAPP initially forms H-bonds with the membrane surface that anchored the protein, and then inserts into the membrane through the hydrophobic interactions between the residues and the hydrophobic tails of the lipids. hIAPP can insert into the membrane more deeply with a larger curvature and with a stronger binding strength. Our result provided important insights into the mechanism of the membrane curvature-dependent property of proteins with molecular details.     

Combined Subcutaneous Fat Aspirate and Skin Tru-Cut Biopsy for Amyloid Screening in Patients with Suspected Systemic Amyloidosis

Screening for systemic amyloidosis is typically carried out with abdominal fat aspirates with varying reported sensitivities. Fat aspirates are preferred for use in primary screening instead of organ biopsies as they are less invasive and thereby minimize the potential risk of complications. At Odense Amyloidosis Center, we performed a prospective study on whether the combined use of fat aspirate and tru-cut skin biopsy could increase the diagnostic sensitivity. Both fat aspirates and skin biopsies were screened with Congo Red staining, and positive biopsies were subsequently subtyped using immunoelectron microscopy and mass spectrometry. Seventy-six patients were included. In total, 24 patients had systemic amyloidosis (11 AL, 12 wtATTR, 1 AA), and 6 patients had localized amyloidosis. Combined fat aspirate and skin biopsy were Congo Red-positive in 15 patients (overall sensitivity (OS) 62.5%). Fat aspirates were positive in 14 patients (OS 58.3%), and the skin biopsy was positive in 5 patients (OS 20.8%). In only one patient did the skin biopsy add extra diagnostic information. The sensitivity differed between AL and ATTR amyloidosis—81.8% and 41.7%, respectively. Using skin biopsy as the only screening method is not recommended.