Mirco Sorci

A universal pathway for amyloid nucleus and precursor formation for insulin

To help identify the etiological agents for amyloid‐related diseases, attention is focused here on the fibrillar precursors, also called oligomers and protofibrils, and on modeling the reaction kinetics of the formation of the amyloid nucleus. Insulin is a favored model for amyloid formation, not only because amyloidosis can be a problem in diabetes, but also because aggregation and fibrillation causes problems during production, storage, and delivery. Small angle neutron scattering (SANS) is used to measure the temporal formation of insulin oligomers in H2O‐ and D2O‐based solvents and obtain consistent evidence of the composition of the insulin nucleus that comprised three dimers or six monomers similar to that recently proposed in the literature. A simple molecular structural model that describes the growth of oligomers under a wide range of environmental conditions is proposed. The model first involves lengthening or end‐on‐end association of dimers to form three‐dimer nuclei, and then exhibits broadening or side‐on‐side association of nuclei. Using different additives to demonstrate their influence on the kinetics of oligomer formation, we showed that, although the time required to form the nucleus was dependent on a specific system, they all followed a universal pathway for nucleus and precursor formation. The methods and analyses presented here provide the first experimental molecular size description of the details of amyloid nucleus formation and subsequent propagation to fibril precursors independent of kinetics. Proteins 2009.

Time‐dependent insulin oligomer reaction pathway prior to fibril formation: Cooling and seeding

The difficulty in identifying the toxic agents in all amyloid‐related diseases is likely due to the complicated kinetics and thermodynamics of the nucleation process and subsequent fibril formation. The slow progression of these diseases suggests that the formation, incorporation, and/or action of toxic agents are possibly rate limiting. Candidate toxic agents include precursors (some at very low concentrations), also called oligomers and protofibrils, and the fibrils. Here, we investigate the kinetic and thermodynamic behavior of human insulin oligomers (imaged by cryo‐EM) under fibril‐forming conditions (pH 1.6 and 65°C) by probing the reaction pathway to insulin fibril formation using two different types of experiments—cooling and seeding—and confirm the validity of the nucleation model and its effect on fibril growth. The results from both the cooling and seeding studies confirm the existence of a time‐changing oligomer reaction process prior to fibril formation that likely involves a rate‐limiting nucleation process followed by structural rearrangements of intermediates (into β‐sheet rich entities) to form oligomers that then form fibrils. The latter structural rearrangement step occurs even in the absence of nuclei (i.e., with added heterologous seeds). Nuclei are formed at the fibrillation conditions (pH 1.6 and 65°C) but are also continuously formed during cooling at pH 1.6 and 25°C. Within the time‐scale of the experiments, only after increasing the temperature to 65°C are the trapped insulin nuclei and resultant structures able to induce the structural rearrangement step and overcome the energy barrier to form fibrils. This delay in fibrillation and accumulation of nuclei at low temperature (25°C) result in a decrease in the mean length of the fibers when placed at 65°C. Fits of an empirical model to the data provide quantitative measures of the delay in the lag‐time during the nucleation process and subsequent reduction in fibril growth rate resulting from the cooling. Also, the seeding experiments, within the time‐scale of the measurements, demonstrate that fibers can initiate fast fibrillation with dissolved insulin (fresh or taken during the lag‐period) but not with other fibers. Qualitatively this is explained with a conjectual free‐energy space plot. Proteins 2009.

Effect of maghemite nanoparticles on insulin amyloid fibril formation: Selective labeling, kinetics, and fibril removal by a magnetic field

Maghemite (gamma-Fe(2)O(3)) magnetic nanoparticles of 15.0 +/- 2.1 nm were formed by nucleation followed by controlled growth of maghemite thin films on gelatin-iron oxide nuclei. Human insulin amyloid fibrils were formed by incubating the monomeric insulin dissolved in aqueous continuous phase at pH 1.6 and 65 degrees C. Magnetic human insulin amyloid fibrils/gamma-Fe(2)O(3) nanoparticle assemblies were prepared by interacting the gamma-Fe(2)O(3) nanoparticles with the insulin amyloid fibrils during or after their formation. The nanoparticles attached selectively to the insulin fibrils in both cases. The kinetics of the insulin fibrillation process in the absence and the presence of the gamma-Fe(2)O(3) nanoparticles was elucidated. The insulin amyloid fibrils/gamma-Fe(2)O(3) nanoparticle assemblies were easily extracted from the aqueous phase via a magnetic field. We hypothesize that this selective extraction approach may also be applicable for the removal of other amyloidogenic proteins that lead to neurodegenerative diseases (e.g., Alzheimers, Parkinsons, Huntingtons, mad cow, and prion diseases) from their continuous phase, e.g. milk, blood, neurological fluid, etc.

Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 μm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynskis equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.

Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability.

Amyloid diseases, including Alzheimers disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.

Detection and reduction of microaggregates in insulin preparations

Insulin is an important biotherapeutic protein, and it is also a model protein used to study amyloid diseases, such as Alzheimers and Parkinsons. The preparation of the protein can lead to small amounts of aggregate in the solution, which in turn may lead to irreproducible in vitro results. Using several pre-treatment methods, we have determined that pH cycling and diafiltration of the insulin removes microaggregates that may be present in the solution. These microaggregates were not detectable with traditional biochemical methods, but using small-angle neutron scattering, we were able to show that pH cycling reduces the radius of gyration of the insulin. Diafiltration removes the aggregates by size and pH cycling dissolves the aggregates by adjusting the pH through the pI of the protein. Pre-treating the insulin with either pH cycling or diafiltration allowed reproducible kinetics of fibrillation for the insulin protein. Microaggregates are a common problem in protein production, formulation, and preparation; here we show that they are the main cause for inconsistent behavior and how pH cycling and diafiltration can mitigate this problem.

A2T and A2V Aβ peptides exhibit different aggregation kinetics, primary nucleation, morphology, structure, and LTP inhibition

The histopathological hallmark of Alzheimers disease (AD) is the aggregation and accumulation of the amyloid beta peptide (Aβ) into misfolded oligomers and fibrils. Here we examine the biophysical properties of a protective Aβ variant against AD, A2T, and a causative mutation, A2T, along with the wild type (WT) peptide. The main finding here is that the A2V native monomer is more stable than both A2T and WT, and this manifests itself in different biophysical behaviors: the kinetics of aggregation, the initial monomer conversion to an aggregation prone state (primary nucleation), the abundances of oligomers, and extended conformations. Aggregation reaction modeling of the conversion kinetics from native monomers to fibrils predicts the enhanced stability of the A2V monomer, while ion mobility spectrometry‐mass spectrometry measures this directly confirming earlier predictions. Additionally, unique morphologies of the A2T aggregates are observed using atomic force microscopy, providing a basis for the reduction in long term potentiation inhibition of hippocampal cells for A2T compared with A2V and the wild type (WT) peptide. The stability difference of the A2V monomer and the difference in aggregate morphology for A2T (both compared with WT) are offered as alternate explanations for their pathological effects. Proteins 2016; 84:488–500.

A multi‐dimensional approach for fractionating proteins using charged membranes

In an effort to increase selectivity among proteins with crossflow ultrafiltration, we offer and demonstrate a comprehensive approach to fractionate proteins of similar molecular weight and relatively close pI values. This multidimensional approach involves optimizing membrane charge type and density together with operating conditions such as precise control of pH, ionic strength, and transmembrane pressure for reduced membrane fouling. Each filtration experiment was performed in cross‐flow configuration for ∼20 min, allowing fast screening for optimal separation as determined by maximum selectivity, Ψ, and purity, P. Using our comprehensive approach for fractionating mixtures RNase A–lysozyme and BSA–hemoglobin, we obtained values of Ψ = 9.1, P = 95.7%, and Ψ = 6.5, P = 62.1%, respectively. Biotechnol. Bioeng. 2013; 110: 1704–1713.

Detection and structural characterization of insulin prefibrilar oligomers using surface enhanced Raman spectroscopy

In vitro fibril formation typically exhibits a lag phase followed by a rapid elongation phase. Soluble prefibrilar oligomers form as multiple assembly states occur during the lag phase and, after forming a nucleus, rapidly propagate into amyloid aggregates and fibrils. The structure and morphology of amyloid fibrils have been extensively characterized over the last decades, while little is known about the structural organization of the prefibrilar oligomers or their multiple assembly states. The main difficulty in structural characterization of prefibrilar aggregates is their low concentration (pmolar) and their continual reactive conversion. Herein we overcome these difficulties by utilizing Surface‐Enhanced Raman Spectroscopy (SERS) with a model amyloid peptide, insulin. SERS is a powerful analytic tool that is able to provide detection of small molecules down to a single‐molecule level. Using SERS we found that during the 3 lag phase before the onset of insulin fibril formation, the amount of insulin oligomers increased more than twice after the first hour of incubation under fibrillation conditions (pH 1.6, 65°C) and then slowly decreased with time. The latter finding is kinetically linked to the conversion of the prefibrilar oligomers into fibril species. This study provides valuable new information about the time‐dependent structural organization of insulin oligomers and demonstrates the power and potential of SERS for detection and structural characterization of biological specimens present at low concentrations.

Tranilast Binds to Aβ Monomers and Promotes Aβ Fibrillation

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimers disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aβ40 monomers with ∼300 μM affinity. Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.