Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation.
Whey protein/pectin edible films were prepared in the presence of transglutaminase and tested as water barrier coatings of both fried doughnuts and french fries as well as of baked food like “taralli” biscuits. Our results demonstrated an undoubted effect of the produced hydrocolloidal films, known to markedly reduce water vapor permeability, in decreasing moisture loss in both doughnuts and french fries when applied before food frying. At the same time, a significant decrease in oil content was observed in the coated fried foods (about 50 % in doughnuts and 25 % in french fries) with respect to both uncoated controls and whey/soy protein-coated samples. No difference was observed between uncoated and coated both doughnuts and french fries with regard to their texture properties and as confirmed by the data from sensory evaluation tests. Furthermore, since the coating by edible films endowed with low water vapor permeability could be useful to prevent moisture absorption by baked foods, we tested the whey protein/pectin film prepared in the presence of transglutaminase, which was also used to coat taralli biscuits. The proposed methodology resulted to be effective to hinder moisture absorption by biscuits during a long storage period, keeping water content constant from 0 to 50 days, thus preventing the food matrix conversion from a glassy state to a rubbery state which is the major cause of baked food rejection by consumers
The aim of this work was to prepare bioplastics, from renewable and biodegradable molecules, to be used as edible films. In particular, grass pea (Lathyrus sativus L.) flour was used as biopolymer source, the proteins of which were structurally modified by means of microbial transglutaminase, an enzyme able to catalyze isopeptide bonds between glutamines and lysines. We analyzed, by means of Zeta-potential, the flour suspension with the aim to determine which pH is more stable for the production of film-forming solutions. The bioplastics were produced by casting and they were characterized according to several technological properties. Optical analysis demonstrated that films cast in the presence of the microbial enzyme are more transparent compared to the untreated ones. Moreover, the visualization by scanning electron microscopy demonstrated that the enzyme-modified films possessed a more compact and homogeneous structure. Furthermore, the presence of microbial transglutaminase allowed to obtain film more mechanically resistant. Finally, digestion experiments under physiological conditions performed in order to obtain information useful for applying these novel biomaterials as carriers in the industrial field, indicated that the enzyme-treated coatings might allow the delivery of bioactive molecules in the gastro-intestinal tract.
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