Jørn Døvling Kaspersen

How Epigallocatechin Gallate Can Inhibit α-Synuclein Oligomer Toxicity in Vitro

Background: Protein oligomers are implicated as cytotoxic membrane-disrupting agents in neurodegenerative diseases. Results: The small molecule EGCG, which inhibits α-synuclein oligomer toxicity, moderately reduces membrane binding and immobilizing the oligomer C-terminal tail. Conclusion: The α-synuclein oligomer may disrupt membranes by vesicle destabilization rather than pore formation. Significance: Limited reduction of oligomer membrane affinity may be sufficient to prevent cytotoxicity. Oligomeric species of various proteins are linked to the pathogenesis of different neurodegenerative disorders. Consequently, there is intense focus on the discovery of novel inhibitors, e.g. small molecules and antibodies, to inhibit the formation and block the toxicity of oligomers. In Parkinson disease, the protein α-synuclein (αSN) forms cytotoxic oligomers. The flavonoid epigallocatechin gallate (EGCG) has previously been shown to redirect the aggregation of αSN monomers and remodel αSN amyloid fibrils into disordered oligomers. Here, we dissect EGCGs mechanism of action. EGCG inhibits the ability of preformed oligomers to permeabilize vesicles and induce cytotoxicity in a rat brain cell line. However, EGCG does not affect oligomer size distribution or secondary structure. Rather, EGCG immobilizes the C-terminal region and moderately reduces the degree of binding of oligomers to membranes. We interpret our data to mean that the oligomer acts by destabilizing the membrane rather than by direct pore formation. This suggests that reduction (but not complete abolition) of the membrane affinity of the oligomer is sufficient to prevent cytotoxicity.

Generic Structures of Cytotoxic Liprotides: Nano‐Sized Complexes with Oleic Acid Cores and Shells of Disordered Proteins

The cytotoxic complex formed between α‐lactalbumin and oleic acid (OA) has inspired many studies on protein–fatty acid complexes, but structural insight remains sparse. After having used small‐angle X‐ray scattering (SAXS) to obtain structural information, we present a new, generic structural model of cytotoxic protein–oleic acid complexes, which we have termed liprotides (lipids and partially denatured proteins). Twelve liprotides formed from seven structurally unrelated proteins and prepared by different procedures all displayed core–shell structures, each with a micellar OA core and a shell consisting of flexible, partially unfolded protein, which stabilizes the OA micelle. The common structure explains similar effects exerted on cells by different liprotides and is consistent with a cargo off‐loading of the OA into cell membranes.

Formation of dynamic soluble surfactant-induced amyloid β peptide aggregation intermediates

Background: β-Structured oligomers of the amyloid β peptide are considered neurotoxic and on-pathway to amyloid fibril formation. Results: Surfactant-induced co-aggregated oligomers show dynamic rapid exchange with free peptide during a slow fibril formation process. Conclusion: β-Structure inducing small molecules kinetically promote peptide assembly into co-aggregates. Significance: Knowledge about molecular mechanisms of peptide aggregation modulators is potentially helpful for therapeutic purposes. Intermediate amyloidogenic states along the amyloid β peptide (Aβ) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aβ aggregation, we here investigate surfactant-induced Aβ aggregation. This process leads to co-aggregates featuring a β-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aβ in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via β-structure to the fully formed α-helical state at high surfactant concentration. The β-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aβ co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (kex ∼1100 s−1) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with β-structure promoting substances, such as surfactants, Aβ is kinetically driven toward an aggregation-prone state.

The role of nanometer-scaled ligand patterns in polyvalent binding by large mannan-binding lectin oligomers

Mannan-binding lectin (MBL) is an important protein of the innate immune system and protects the body against infection through opsonization and activation of the complement system on surfaces with an appropriate presentation of carbohydrate ligands. The quaternary structure of human MBL is built from oligomerization of structural units into polydisperse complexes typically with three to eight structural units, each containing three lectin domains. Insight into the connection between the structure and ligand-binding properties of these oligomers has been lacking. In this article, we present an analysis of the binding to neoglycoprotein-coated surfaces by size-fractionated human MBL oligomers studied with small-angle x-ray scattering and surface plasmon resonance spectroscopy. The MBL oligomers bound to these surfaces mainly in two modes, with dissociation constants in the micro to nanomolar order. The binding kinetics were markedly influenced by both the density of ligands and the number of ligand-binding domains in the oligomers. These findings demonstrated that the MBL-binding kinetics are critically dependent on structural characteristics on the nanometer scale, both with regard to the dimensions of the oligomer, as well as the ligand presentation on surfaces. Therefore, our work suggested that the surface binding of MBL involves recognition of patterns with dimensions on the order of 10–20 nm. The recent understanding that the surfaces of many microbes are organized with structural features on the nanometer scale suggests that these properties of MBL ligand recognition potentially constitute an important part of the pattern-recognition ability of these polyvalent oligomers.

Cooperative binding of LysM domains determines the carbohydrate affinity of a bacterial endopeptidase protein

Cellulose, chitin and peptidoglycan are major long‐chain carbohydrates in living organisms, and constitute a substantial fraction of the biomass. Characterization of the biochemical basis of dynamic changes and degradation of these β,1–4‐linked carbohydrates is therefore important for both functional studies of biological polymers and biotechnology. Here, we investigated the functional role of multiplicity of the carbohydrate‐binding lysin motif (LysM) domain that is found in proteins involved in bacterial peptidoglycan synthesis and remodelling. The Bacillus subtilis peptidoglycan‐hydrolysing NlpC/P60 d,l‐endopeptidase, cell wall‐lytic enzyme associated with cell separation, possesses four LysM domains. The contribution of each LysM domain was determined by direct carbohydrate‐binding studies in aqueous solution with microscale thermophoresis. We found that bacterial LysM domains have affinity for N‐acetylglucosamine (GlcNac) polymers in the lower‐micromolar range. Moreover, we demonstrated that a single LysM domain is able to bind carbohydrate ligands, and that LysM domains act additively to increase the binding affinity. Our study reveals that affinity for GlcNAc polymers correlates with the chain length of the carbohydrate, and suggests that binding of long carbohydrates is mediated by LysM domain cooperativity. We also show that bacterial LysM domains, in contrast to plant LysM domains, do not discriminate between GlcNAc polymers, and recognize both peptidoglycan fragments and chitin polymers with similar affinity. Finally, an Ala replacement study suggested that the carbohydrate‐binding site in LysM‐containing proteins is conserved across phyla.

Low-Resolution Structures of OmpA⋅DDM Protein–Detergent Complexes

We have used SAXS to determine the low‐resolution structure of the outer‐membrane protein OmpA from E. coli solubilized by the surfactant dodecyl maltoside (DDM). We have studied three variants of the transmembrane domain of OmpA—namely monomers, self‐associated dimers, and covalently linked dimers—as well as the monomeric species of the full‐length protein with the periplasmic domain. We can successfully model the structures of the monomeric and covalently linked dimer as one and two natively folded proteins in a DDM micelle, respectively, whereas the noncovalently linked dimer presents a more complicated structure, possibly due to higher‐order species. We have determined the structure of the full‐length protein to be that of a globular periplasmic domain attached through a flexible linker to the transmembrane domain. This approach provides valuable information about how membrane proteins are embedded in amphiphilic environments.

Refolding of SDS-Unfolded Proteins by Nonionic Surfactants

The strong and usually denaturing interaction between anionic surfactants (AS) and proteins/enzymes has both benefits and drawbacks: for example, it is put to good use in electrophoretic mass determinations but limits enzyme efficiency in detergent formulations. Therefore, studies of the interactions between proteins and AS as well as nonionic surfactants (NIS) are of both basic and applied relevance. The AS sodium dodecyl sulfate (SDS) denatures and unfolds globular proteins under most conditions. In contrast, NIS such as octaethylene glycol monododecyl ether (C12E8) and dodecyl maltoside (DDM) protect bovine serum albumin (BSA) from unfolding in SDS. Membrane proteins denatured in SDS can also be refolded by addition of NIS. Here, we investigate whether globular proteins unfolded by SDS can be refolded upon addition of C12E8 and DDM. Four proteins, BSA, α-lactalbumin (αLA), lysozyme, and β-lactoglobulin (βLG), were studied by small-angle x-ray scattering and both near- and far-UV circular dichroism. All proteins and their complexes with SDS were attempted to be refolded by the addition of C12E8, while DDM was additionally added to SDS-denatured αLA and βLG. Except for αLA, the proteins did not interact with NIS alone. For all proteins, the addition of NIS to the protein-SDS samples resulted in extraction of the SDS from the protein-SDS complexes and refolding of βLG, BSA, and lysozyme, while αLA changed to its NIS-bound state instead of the native state. We conclude that NIS competes with globular proteins for association with SDS, making it possible to release and refold SDS-denatured proteins by adding sufficient amounts of NIS, unless the protein also interacts with NIS alone.

Glycolipid Biosurfactants Activate, Dimerize, and Stabilize Thermomyces lanuginosus Lipase in a pH-Dependent Fashion

We present a study of the interactions between the lipase from Thermomyces lanuginosus (TlL) and the two microbially produced biosurfactants (BSs), rhamnolipid (RL) and sophorolipid (SL). Both RL and SL are glycolipids; however, RL is anionic, while SL is a mixture of anionic and non-ionic species. We investigate the interactions of RL and SL with TlL at pH 6 and 8 and observe different effects at the two pH values. At pH 8, neither RL nor SL had any major effect on TlL stability or activity. At pH 6, in contrast, both surfactants increase TlLs thermal stability and fluorescence and activity measurements indicate interfacial activation of TlL, resulting in 3- and 6-fold improved activity in SL and RL, respectively. Nevertheless, isothermal titration calorimetry reveals binding of only a few BS molecules per lipase. Size-exclusion chromatography and small-angle X-ray scattering suggest formation of TlL dimers with binding of small amounts of either RL or SL at the dimeric interface, forming an elongated complex. We conclude that RL and SL are compatible with TlL and constitute promising green alternatives to traditional surfactants.

Promoting protein self-association in non-glycosylated Thermomyces lanuginosus lipase based on crystal lattice contacts.

We have used the crystal structure of Thermomyces lanuginosus lipase (TlL) to identify and strengthen potential protein-protein interaction sites in solution. As wildtype we used a deglycosylated mutant of TlL (N33Q). We designed a number of TlL mutants to promote interactions via interfaces detected in the crystal-lattice structure, through strengthening of hydrophobic, polar or electrostatic contacts or truncation of sterically blocking residues. We identify a mutant predicted to lead to increased interfacial hydrophobic contacts (N92F) that shows markedly increased self-association properties on native gradient gels. While wildtype TlL mainly forms monomer and <5% dimers, N92F forms stable trimers and dimers according to Size-Exclusion Chromatography and Small-Angle X-ray Scattering. These oligomers account for ~25% of the population and their enzymatic activity is comparable to that of the monomer. Self-association stabilizes TlL against thermal denaturation. Furthermore, the trimer is stable to dilution and requires high concentrations (>2M) of urea to dissociate. We conclude that crystal lattice contacts are a good starting point for design strategies to promote protein self-association.

Formation of Dynamic Soluble Surfactant-Induced Amyloid Beta Peptide Aggregation Intermediates

The 40-42 residue Amyloid β (Aβ) peptide forms β-structured oligomers on-pathway to amyloid fibril formation and are linked to neuronal damage characteristic for Alzheimers disease. Surfactants such as SDS induce relatively stable β-structured Aβ co-aggregates and may be considered as a model system for lipids. We have characterized different intermediate aggregation states appearing during the Aβ aggregation process, as well as the kinetics of their formation and dynamic exchange between free and bound peptide. A broad range of biophysical techniques were used, including small angle X-ray scattering (SAXS) and NMR spectroscopy, particularly 15N-CPMG relaxation dispersion experiments.Aβ shows a three-state secondary structure transition depending on surfactant concentration, from random coil-like, via β-structure to α-helix at high surfactant concentration. Structural information on the β-structured co-aggregates was obtained by SAXS experiments that at the beginning showed a large fraction of globular co-aggregates (diameter ∼ 75 A). This fraction gradually vanished on a min to hr timescale and elongated co-aggregated fibrils were formed (diameter ∼ 60 A and length > 350 A), in line with transmission electron microscopy images. A fast dynamic exchange process (kex ∼ 1100 s-1) between free and co-aggregate bound peptide takes place, as monitored by NMR relaxation dispersion experiments.This study of the surfactant-induced Aβ aggregation may serve as a model for aggregation of Aβ alone or in the presence of lipids.Reference:1. Abelein, A. et al., J. Biol. Chem. (2013), 288, 23518-23528.