The apomyoglobin mutant W7FW14F forms amyloidlike fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing -structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.
Myoglobin is an ␣-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of -structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.Such chronic disorders as Alzheimer's disease, senile systemic amyloidosis, transmissible spongiform encephalopathies, and dialysis-related amyloidosis are characterized by the extracellular deposition of insoluble protein aggregates known as amyloid fibrils (1-6). About 20 proteins are now known to be involved in the generation of amyloid in vivo. Fibril formation is initiated in vitro under conditions that stabilize partially unfolded soluble intermediates of the native proteins either after the partial destabilization of physiologically folded proteins in the case of globular proteins (7) or after the partial stabilization (i.e. folding) of random coil polypeptide chains in the case of natively unfolded proteins (8). Despite substantial differences in both sequence and length (from 40 to 250 residues), all the proteins responsible for amyloid deposition form fibrils composed of -strands oriented perpendicularly to the long axis of the fibril (9). Electron microscopy shows that the fibrils are straight and unbranched and are 40 -120 Å in diameter (9, 10). Also proteins not known to be associated with amyloid disease may form amyloid fibrils under in vitro conditions that favor partially folded states (11-15). These states are more prone to aggregation than the native state because hydrophobic residues, which are largely buried within the core of the native protein, become more exposed upon partial unfolding. The way in which proteins aggregate in the test tube is remarkably similar to how proteins form the so-called "amyloid" deposits. Even myoglobin, an ordinary all-␣ globular protein, can form fibrils containing -strands under experimental conditions that favor the formation of partially folded states (15). Thus, amyloid formation d...
The main objective of this work was to obtain microencapsulated probiotic cells in order to improve their resistance to heat stress and gastrointestinal conditions. A further aim was to obtain a potentially probiotic chocolate soufflé. Lactobacillus reuteri DSM 17938 cells were microencapsulated by spray drying in alginate matrix and further coated with chitosan. Bacterial survival after exposure to different heat treatments and simulated gastrointestinal conditions were measured to test the microcapsules. They were also dyed by using a LIVE/DEAD® BacLight™ Bacterial Viability Kit and characterized by epifluorescence microscope observation. Furthermore, a potentially chocolate soufflé was prepared using microencapsulated cells. The results indicated that alginate microcapsules did not improve acid tolerance or heat resistance in “in vitro” experiments, while they were able to protect 7% of the Lactobacillus reuteri population during the baking of a chocolate soufflé, compared to a survival rate of 1% of free cells. By contrast, the cells microencapsulated with alginate coated with chitosan showed, compared to free cells, improved acid tolerance, allowing the cell population to remain constant after 3 h in simulated gastric conditions. Moreover, the heat resistance of cells in co-cross-linked microcapsules significantly improved compared to free cells, both in “in vitro” and “in food” experiments. Microencapsulation led to a survival rate of 10% after baking a chocolate soufflé. However, the final level of bacterial cells in the product was too low to consider the chocolate soufflé as a probiotic product
A significant number of fatal diseases are classified as protein deposition disorders, in which a normally soluble protein is deposited in an insoluble amyloid form. It has been reported that tetracycline exhibits anti-amyloidogenic activity by inhibiting aggregate formation and disaggregating preformed fibrils. In this work, we examined the effect induced by the presence of tetracycline on the fibrillogenesis and cytotoxicity of the amyloid-forming apomyoglobin mutant W7FW14F. Like other amyloid-forming proteins, early prefibrillar aggregates formed by this protein are highly cytotoxic, whereas insoluble mature fibrils are not. The effect induced by tetracycline on the fibrillation process has been examined by atomic force microscopy, light scattering, DPH staining, and thioflavin T fluorescence. The cytotoxicity of the amyloid aggregates was estimated by measuring cell viability using MTT assay. The results show that tetracycline acts as anti-aggregating agent, which inhibits the fibril elongation process but not the early aggregation steps leading to the formation of soluble oligomeric aggregates. Thus, this inhibition keeps the W7FW14F mutant in a prefibrillar, highly cytotoxic state. In this respect, a careful usage of tetracycline as fibril inhibitor is indicated.
The conformational properties of partially folded states of apomyoglobin have been investigated using an integrated approach based on fluorescence spectroscopy and hydrogen/deuterium exchange followed by mass spectrometry. The examined states were those obtained: (i) by adding 4% v/v hexafluoroisopropanol to native myoglobin, HFIP-MG(N); (ii) by adding 4% v/v hexafluoroisopropanol to acid unfolded myoglobin, HFIP-MG(U); (iii) at pH 3.8, I-1 state; and (iv) at pH 2.0-0.2 M NaCl, A state. Proteolytic digestion of the hydrogen/deuterium exchanged proteins showed that, in I-1 state, the helices C, D, E, and F incorporate more deuterium, whereas in HFIP-MG(N) the exchange rate is similar for all protein regions. These results suggest that I-1 contains the ABGH domain in a native-like organization, whereas HFIP-MG(N) loses a large number of tertiary interactions, thus acquiring a more flexible structure. The fluorescence data are consistent with the above picture. In fact, the tryptophan/ANS energy transfer is much less efficient for the ANS-HFIP-MG(N) complex than for the other complexes, thus suggesting that the distances between the fluorophores might be increased. Moreover, fluorescence polarization measurements indicated that the rotational motion of HFIP-MG(N) occurs on a longer time scale than the other partially folded states, thus suggesting that the volume of this state could be larger. The overall results indicate that addition of hexafluoroisopropanol to native myoglobin results in the formation of a true molten globule where tertiary interactions are reduced, while the secondary structure and the globular compactness are conserved.
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