Kerensa Broersen

The mechanism of γ-Secretase dysfunction in familial Alzheimer disease.

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid β (Aβ)42 relative to Aβ40 by an unknown, possibly gain‐of‐toxic‐function, mechanism. However, many PSEN mutations paradoxically impair γ‐secretase and ‘loss‐of‐function’ mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aβ generation via three different mechanisms, resulting in qualitative changes in the Aβ profiles, which are not limited to Aβ42. Loss of ε‐cleavage function is not generally observed among FAD mutants. On the other hand, γ‐secretase inhibitors used in the clinic appear to block the initial ε‐cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase‐like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aβ products, and suggest fundamental improvements for current drug development efforts.

Structural Basis for Increased Toxicity of Pathological Aβ42:Aβ40 Ratios in Alzheimer Disease

Background: Amyloid β peptide plays a role in Alzheimer disease. Results: Interaction of amyloid β peptides with 40 and 42 amino acids has consequences for oligomer formation. Conclusion: Increased production of amyloid β peptide with 42 amino acids affects the behavior of the entire amyloid β peptide pool. Significance: This might explain the synaptotoxic effect observed with a shift in amyloid β peptide production. The β-amyloid peptide (Aβ) is directly related to neurotoxicity in Alzheimer disease (AD). The two most abundant alloforms of the peptide co-exist under normal physiological conditions in the brain in an Aβ42:Aβ40 ratio of ∼1:9. This ratio is often shifted to a higher percentage of Aβ42 in brains of patients with familial AD and this has recently been shown to lead to increased synaptotoxicity. The molecular basis for this phenomenon is unclear. Although the aggregation characteristics of Aβ40 and Aβ42 individually are well established, little is known about the properties of mixtures. We have explored the biophysical and structural properties of physiologically relevant Aβ42:Aβ40 ratios by several techniques. We show that Aβ40 and Aβ42 directly interact as well as modify the behavior of the other. The structures of monomeric and fibrillar assemblies formed from Aβ40 and Aβ42 mixtures do not differ from those formed from either of these peptides alone. Instead, the co-assembly of Aβ40 and Aβ42 influences the aggregation kinetics by altering the pattern of oligomer formation as evidenced by a unique combination of solution nuclear magnetic resonance spectroscopy, high molecular weight mass spectrometry, and cross-seeding experiments. We relate these observations to the observed enhanced toxicity of relevant ratios of Aβ42:Aβ40 in synaptotoxicity assays and in AD patients.

Transient dynamics of Aβ contribute to toxicity in Alzheimer's disease.

The aggregation and deposition of the amyloid-β peptide (Aβ) in the brain has been linked with neuronal death, which progresses in the diagnostic and pathological signs of Alzheimer’s disease (AD). The transition of an unstructured monomeric peptide into self-assembled and more structured aggregates is the crucial conversion from what appears to be a harmless polypeptide into a malignant form that causes synaptotoxicity and neuronal cell death. Despite efforts to identify the toxic form of Aβ, the development of effective treatments for AD is still limited by the highly transient and dynamic nature of interconverting forms of Aβ. The variability within the in vivo “pool” of different Aβ peptides is another complicating factor. Here we review the dynamical interplay between various components that influence the heterogeneous Aβ system, from intramolecular Aβ flexibility to intermolecular dynamics between various Aβ alloforms and external factors. The complex dynamics of Aβ contributes to the causative role of Aβ in the pathogenesis of AD.

A standardized and biocompatible preparation of aggregate-free amyloid beta peptide for biophysical and biological studies of Alzheimer's disease

We provide a validated and rapid protocol for the solubilization of amyloid β-peptide (Aβ). This procedure involves sequential solubilization using structure-breaking organic solvents hexafluoroisopropanol and DMSO followed by column purification. The low solubility and tendency of Aβ to aggregate considerably impede the in vitro handling and biophysical or biological investigation of Aβ, despite the interest in this peptide because of its implication in Alzheimers disease. The main advantage of the proposed protocol over others is that it results in standardized aggregate-free Aβ peptide samples that are biocompatible for cell culture studies and yield reproducible aggregation kinetics and cytotoxicities. This three-step protocol also enables the co-solubilization of the longer Aβ42 variant with Aβ40 in ratios relevant to Alzheimers disease.

A comparative analysis of the aggregation behavior of amyloid-β peptide variants

Amyloid beta and Amyloid beta bind by filter binding (View Interaction: 1, 2, 3)

Mechanistic insights in glycation-induced protein aggregation.

Protein glycation causes loss-of-function through a process that has been associated with several diabetic-related diseases. Additionally, glycation has been hypothesized as a promoter of protein aggregation, which could explain the observed link between hyperglycaemia and the development of several aggregating diseases. Despite its relevance in a range of diseases, the mechanism through which glycation induces aggregation remains unknown. Here we describe the molecular basis of how glycation is linked to aggregation by applying a variety of complementary techniques to study the nonenzymatic glycation of hen lysozyme with ribose (ribosylation) as the reducing carbohydrate. Ribosylation involves a chemical multistep conversion that induces chemical modifications on lysine side chains without altering the protein structure, but changing the protein charge and enlarging its hydrophobic surface. These features trigger lysozyme native-like aggregation by forming small oligomers that evolve into bigger insoluble particles. Moreover, lysozyme incubated with ribose reduces the viability of SH-SY5Y neuroblastoma cells. Our new insights contribute toward a better understanding of the link between glycation and aggregation.

Optimized triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

Scope Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for β-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays. Methods and results Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination. Conclusion The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

New mimetic peptides inhibitors of Αβ aggregation. Molecular guidance for rational drug design

A new series of mimetic peptides possessing a significant Aβ aggregation modulating effect was reported here. These compounds were obtained based on a molecular modelling study which allowed us to perform a structural-based virtual selection. Monitoring Aβ aggregation by thioflavin T fluorescence and transmission electron microscopy revealed that fibril formation was significantly decreased upon prolonged incubation in presence of the active compounds. Dot blot analysis suggested a decrease of soluble oligomers strongly associated with cognitive decline in Alzheimers disease. For the molecular dynamics simulations, we used an Aβ42 pentameric model where the compounds were docked using a blind docking technique. To analyze the dynamic behaviour of the complexes, extensive molecular dynamics simulations were carried out in explicit water. We also measured parameters or descriptors that allowed us to quantify the effect of these compounds as potential inhibitors of Aβ aggregation. Thus, significant alterations in the structure of our Aβ42 protofibril model were identified. Among others we observed the destruction of the regular helical twist, the loss of a stabilizing salt bridge and the loss of a stabilizing hydrophobic interaction in the β1 region. Our results may be helpful in the structural identification and understanding of the minimum structural requirements for these molecules and might provide a guide in the design of new aggregation modulating ligands.

Application Potential of Food Protein Modification

Proteins are essential in foods, not only for their nutritional value, but also as modulator of structure and perception of a food product. The functional behavior of a protein is inherently susceptible to physico-chemical conditions as pH, ionic strength, temperature, or pressure, making them also an unpredictable, and at the same time, opportune component in food production. Proteins are generally also industrially costly, and with increasing world population and welfare the pressure on protein-availability for food purposes gives rise to some concerns. In view of a more sustainable use of protein-sources a number of routes have been followed in the past decades that provided big steps forward in protein availability: (i) more efficient production or protein refinery methods, (ii) use of alternative protein sources, and (iii) optimized usage of protein functionality. Especially in wheat production correlations between genetic expression and functional product behavior allowed breeders to optimize cultivars for geographic location (e.g. Payne et al., 1984). Alternatively, one has the ability to express specific proteins in non-original sources, for example human milk proteins in plants, such as rice (e.g. Lonnerdal, 2002). Directed alterations in the genome of food-producing organisms can lead to changes in the primary sequences of relevant proteins and thereby introduce potentially new functionality. If sufficient quantities of the novel protein are synthesized and become admixed with the basal levels of protein in the food, the functional properties of the food system (textureformation) may become improved. Alternatively, the modified protein can be isolated for use as food ingredient. More recently, a number of proteins from less-conventional origin have been identified as human food ingredients that one has started to exploit, e.g. algae, leafs, insects, and various seeds. Successful utilization of these new proteinaceous materials has thus far been rather limited, requiring breakthroughs in extractability, their digestibility, nutritive value, and overall functional and organoleptic properties. More downstream in the process is the modulation of protein functional behavior at an ingredient level. This can be physical-chemically, enzymatically, or via chemical engineering.

Two distinct β-sheet structures in Italian-mutant amyloid-beta fibrils: A potential link to different clinical phenotypes

Most Alzheimer’s disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aβ) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aβ mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased β-amyloid accumulation, and Aβ deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aβ1–42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aβ both form fibrils characterized by the cross-β architecture, but with distinct β-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aβ1–42 oligomers and fibrils both display an antiparallel β-sheet structure, in comparison with the parallel β-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aβ fibrils in a pH-dependent manner, in terms of their underlying β-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel β-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation.