Erik H. Nielsen

Coexistence of ribbon and helical fibrils originating from hIAPP(20-29) revealed by quantitative nanomechanical atomic force microscopy.

Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP20–29, which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP20–29 nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP20–29 shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing β sheets in cross-β motifs as the basis of self-assembled amyloids.

Long-Term-Stable Ether−Lipid vs Conventional Ester−Lipid Bicelles in Oriented Solid-State NMR: Altered Structural Information in Studies of Antimicrobial Peptides

Recently, ether lipids have been introduced as long-term stable alternatives to the more natural, albeit easier degradable, ester lipids in the preparation of oriented lipid bilayers and bicelles for oriented-sample solid-state NMR spectroscopy. Here we report that ether lipids such as the frequently used 14-O-PC (1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine) may induce significant changes in the structure and dynamics, including altered interaction between peptides and lipids relative to what is observed with the more conventionally used DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers. Such effects are demonstrated for the antimicrobial peptide novicidin, for which 2D separate-local-field NMR and circular dichroism experiments reveal significant structural/conformational differences for the peptide in the two different lipid systems. Likewise, we observe altered secondary structure and different temperature-dependent membrane anchoring for the antimicrobial peptide alamethicin depending on whether the peptide is reconstituted into ester or ether lipids. Such observations are not particularly surprising considering the significant difference of the lipids in the phosphorus headgroup and they may provide important new insight into the delicate peptide-membrane interactions in the systems studied. In contrast, these observations reinforce the need to carefully consider potential structural changes in addition to long-term stability prior to the selection of membrane environment of membrane proteins in the analysis of their structure and dynamics. In more general terms, the results underscore the necessity in structural biology to address both the protein and its environments in studies relating structure to function.

The importance of being capped: Terminal capping of an amyloidogenic peptide affects fibrillation propensity and fibril morphology.

The formation of aggregated fibrillar β-sheet structures has been proposed to be a generic feature of proteins. Aggregation propensity is highly sequence dependent, and often only part of the protein is incorporated into the fibril core. Therefore, shorter peptide fragments corresponding to the fibril core are attractive fibrillation models. The use of peptide models introduces new termini into the fibrils, yet little attention has been paid to the role these termini may play in fibrillation. Here, we report that terminal modifications of a 10-residue peptide fragment of human islet amyloid polypeptide strongly affect fibrillation kinetics and the resulting fibril morphology. Capping of the N-terminus abolishes fibrillation, while C-terminal capping results in fibrils with a twisted morphology. Peptides with either both termini free or both termini capped form flat fibrils. Molecular dynamics simulations reveal that the N-terminal acetyl cap folds up and interacts with the peptides hydrophobic side chains, while the uncapped N-terminus in the C-terminally capped version results in twisting of the fibrils due to charge repulsion from the free N-termini. Our results highlight the role of terminal interactions in fibrillation of small peptides and provide molecular insight into the consequences of C-terminal modifications frequently found in peptide hormones in vivo.

Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone.

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.

Scanning Tunneling Microscopy Reveals Single-Molecule Insights into the Self-Assembly of Amyloid Fibrils

Many severe diseases are associated with amyloid fibril deposits in the body caused by protein misfolding. Structural information on amyloid fibrils is accumulating rapidly, but little is known about the assembly of peptides into fibrils at the level of individual molecules. Here we investigate self-assembly of the fibril-forming tetrapeptides KFFE and KVVE on a gold surface under ultraclean vacuum conditions using scanning tunneling microscopy. Combined with restrained molecular dynamics modeling, we identify peptide arrangements with interesting similarities to fibril structures. By resolving individual peptide residues and revealing conformational heterogeneities and dynamics, we demonstrate how conformational correlations may be involved in cooperative fibril growth. Most interestingly, intermolecular interactions prevail over intramolecular interactions, and assembly of the phenyl-rich KFFE peptide appears not to be dominated by π-π interactions. This study offers interesting perspectives for obtaining fundamental single-molecule insights into fibril formation using a surface science approach to study idealized model systems.

How Glycosaminoglycans Promote Fibrillation of Salmon Calcitonin

Glycosaminoglycans (GAGs) bind all known amyloid plaques and help store protein hormones in (acidic) granular vesicles, but the molecular mechanisms underlying these important effects are unclear. Here we investigate GAG interactions with the peptide hormone salmon calcitonin (sCT). GAGs induce fast sCT fibrillation at acidic pH and only bind monomeric sCT at acidic pH, inducing sCT helicity. Increasing GAG sulfation expands the pH range for binding. Heparin, the most highly sulfated GAG, binds sCT in the pH interval 3–7. Small angle x-ray scattering indicates that sCT monomers densely decorate and pack single heparin chains, possibly via hydrophobic patches on helical sCT. sCT fibrillates without GAGs, but heparin binding accelerates the process by decreasing the otherwise long fibrillation lag times at low pH and accelerates fibril growth rates at neutral pH. sCT·heparin complexes form β-sheet-rich heparin-covered fibrils. Solid-state NMR reveals that heparin does not alter the sCT fibrillary core around Lys11 but makes changes to Val8 on the exterior side of the β-strand, possibly through contacts to Lys18. Thus GAGs significantly modulate sCT fibrillation in a pH-dependent manner by interacting with both monomeric and aggregated sCT.

Scaffolded multimers of hIAPP20–29 peptide fragments fibrillate faster and lead to different fibrils compared to the free hIAPP20–29 peptide fragment

Applying fibril-forming peptides in nanomaterial design is still challenged by the difficulties in understanding and controlling how fibrils form. The present work investigates the influence of motional restriction on peptide fibrillation. We use cyclotriphosphazene and cyclodextrin as templates to make conjugates of the fibril-forming core of human islet amyloid polypeptide. Attachment of the peptide to the templates resulted in multimers containing six peptide fragments at different positions. ThT fluorescence, CD and FTIR spectroscopy, and AFM and TEM imaging reveal that in both conjugates the peptide retained its fibrillating properties and formed fibrils. However, the conjugate fibrils formed more rapidly than the free peptide and were long and thin, as opposed to the thick and twisted morphology of the intact peptide. Thus the motional restrictions introduced by the scaffold modulate the structure of the fibrils but do not impede the actual fibrillation process.

Measurements of gas pressure in voids in epoxy castings for high voltage equipment

An investigation of samples of epoxy each containing one void, which were produced at different pressures, is reported. The samples were of the disk type with the void located in the center. The gas in the voids has a pressure somewhat related to the curing pressure, thereby directly influencing the partial-discharge inception voltage. Data show that gas pressure in voids in epoxy castings can be determined by use of an ultrasound test method. A relationship between the void gas pressure and the epoxy curing pressure is also found. This investigation is part of an effort to predict the inception voltage of partial discharges in high-voltage epoxy castings from the processing parameters.<<ETX>>

Nigrostriatal proteasome inhibition impairs dopamine neurotransmission and motor function in minipigs

ABSTRACT Parkinsons disease (PD) is characterized by degeneration of dopaminergic neurons in the substantia nigra leading to slowness and stiffness of limb movement with rest tremor. Using ubiquitin proteasome system inhibitors, rodent models have shown nigrostriatal degeneration and motor impairment. We translated this model to the Göttingen minipig by administering lactacystin into the medial forebrain bundle (MFB). Minipigs underwent positron emission tomography (PET) imaging with (+)‐&agr;‐[11C]dihydrotetrabenazine ([11C]DTBZ), a marker of vesicular monoamine transporter 2 availability, at baseline and three weeks after the unilateral administration of 100&mgr;g lactacystin into the MFB. Compared to their baseline values, minipigs injected with lactacystin showed on average a 36% decrease in ipsilateral striatal binding potential corresponding to impaired presynaptic dopamine terminals. Behaviourally, minipigs displayed asymmetrical motor disability with spontaneous rotations in one of the animals. Immunoreactivity for tyrosine hydroxylase (TH) and HLA‐DR‐positive microglia confirmed asymmetrical reduction in nigral TH‐positive neurons with an inflammatory response in the lactacystin‐injected minipigs. In conclusion, direct injection of lactacystin into the MFB of minipigs provides a model of PD with reduced dopamine neurotransmission, TH‐positive neuron reduction, microglial activation and behavioural deficits. This large animal model could be useful in studies of symptomatic and neuroprotective therapies with translatability to human PD. HIGHLIGHTSProteasome inhibition model of Parkinsons disease translated to the minipig.Reduced dopamine neurotransmission observed using PET imaging.Immunohistochemistry showed TH‐positive neuron reduction and microglial activation.Lesioned minipigs displayed compromised motility and coordination.Minipig model of parkinsonism may be useful in future testing of treatments.