UVB pretreatment of ß-lactoglobulin affects the temperature-induced formation of functional amyloid-like aggregates and promotes oxidative degradation.

Unfolding in combination with or without acid hydrolysis is crucial for the formation of functional amyloid (fibrillar) or amyloid-like (worm-like) ß-lactoglobulin (BLG) aggregates, which can be induced through temperature treatment for several hours at pH 2-4. A preceding conformational destabilization of BLG might affect its aggregation. We investigated ultraviolet (UV) B radiation as conformational perturbing treatment to facilitate temperature-induced protein aggregation. 2-h UVB pretreated BLG (UV-BLG) exhibited an accelerated worm-like aggregation at pH 3.5, while at pH 2 the formation of fibrils was decelerated. The UV-induced conformational destabilization lowered the thermal stability and thus facilitates unfolding during thermal treatment. Thereby, the formation of covalent and non-covalent intermolecular interactions was favored, which promoted assembly of intact proteins resulting in worm-like aggregates. The oxidative degradation of UV-BLG was suggested to alter fibrillation-prone protein regions and thereby impede peptide assembly.

Toxic Metamorphosis-How Changes from Lysosomal to Cytosolic pH Modify the Alpha-Synuclein Aggregation Pattern.

Alpha-synuclein (aSyn) is a cytosolic, aggregation-prone protein that is associated with neurodegenerative disorders like Parkinson's disease. Interestingly, the protein can appear in different conformations, including monomeric and oligomeric forms as well as amyloid fibrils. Its individual structural constituents seem to be dependent on various factors and the composition of the respective cellular surroundings. Although under physiological conditions, most aSyn is found in the cytosol and synapses of neurons, aSyn can also be found in lysosomal compartments, where it gets degraded. We here compare the assembly speed, morphology, folding state, and spreading of aSyn at cytosolic pH (pH 7.4) and lysosomal pH (pH 5) using Thioflavin T, transmission electron microscopy, circular dichroism, and Fourier transform infrared spectroscopy. Interestingly, we found substantial differences between aSyn aggregation under neutral and acidic pH conditions, like those present in cytosolic and lysosomal cellular compartments. Also, lysosomal aSyn enriched from an aSyn-overexpressing cell line was able to seed aggregation in a concentration-dependent manner. Moreover, we observed that aSyn aggregates formed under in vitro lysosomal pH (pH 5) conditions were not stable at neutral pH and collapsed into partly soluble aggregates with changed structural characteristics. Our findings have meaningful implications in intracellular toxicity events as well as in lysis procedures for molecular and structural characterization of intracellular aSyn conformers.

Measurement of length distribution of beta-lactoglobulin fibrils by multiwavelength analytical ultracentrifugation.

The whey protein beta-lactoglobulin is the building block of amyloid fibrils which exhibit a great potential in various applications. These include stabilization of gels or emulsions. During biotechnological processing, high shear forces lead to fragmentation of fibrils and therefore to smaller fibril lengths. To provide insight into such processes, pure straight amyloid fibril dispersions (prepared at pH 2) were produced and sheared using the rotor stator setup of an Ultra Turrax. In the first part of this work, the sedimentation properties of fragmented amyloid fibrils sheared at different stress levels were analyzed with mulitwavelength analytical ultracentrifugation (AUC). Sedimentation data analysis was carried out with the boundary condition that fragmented fibrils were of cylindrical shape, for which frictional properties are known. These results were compared with complementary atomic force microscopy (AFM) measurements. We demonstrate how the sedimentation coefficient distribution from AUC experiments is influenced by the underlying length and diameter distribution of amyloid fibrils.In the second part of this work, we show how to correlate the fibril size reduction kinetics with the applied rotor revolution and the resulting energy density, respectively, using modal values of the sedimentation coefficients obtained from AUC. Remarkably, the determined scaling laws for the size reduction are in agreement with the results for other material systems, such as emulsification processes or the size reduction of graphene oxide sheets.

Protein oxidation during temperature-induced amyloid aggregation of beta-lactoglobulin.

Although the connection between protein oxidation, amyloid aggregation and diseases such as Alzheimer's is well known there is no information on such effects during preparation of beta-lactoglobulin fibrils. Different morphologies of amyloid aggregates of beta-lactoglobulin were prepared by incubation at pH 2 or pH 3.5 for up to 72 h. After 5 h, amyloid aggregates at pH 2 formed typical fibrils, which consisted of peptides. At pH 3.5, the amyloid aggregates were worm-like and consisted of intact protein. After 72 h, the building blocks at both pH values changed towards smaller peptides. The apparent tyrosine oxidation reached a maximum after 5 h at both pH values, whereas N-formylkynurenine and carbonyls increased continuously during 72 h. In case amyloid structures are used as edible material, the health related effects caused by protein oxidation needs to be considered.