Protein N-Homocysteinylation Induces the Formation of Toxic Amyloid-Like Protofibrils |
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Authors: | Paolo Paoli Francesca Sbrana Anna Caselli Paolo Cirri Lucia Formigli Giampaolo Manao Giampietro Ramponi |
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Institution: | 1 Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze, Viale Morgagni 50, 50134 Firenze, Italy 2 Centro per lo Studio delle Dinamiche Complesse, Dipartimento di Fisica, Università degli Studi di Firenze, via Sansone 1, 50019, Sesto F.no, Firenze, Italy 3 Istituto dei Sistemi Complessi—Istituto Nazionale delle Ricerche, via Madonna del piano 10, 50019, Sesto F.no, Firenze, Italy 4 Dipartimento di Anatomia, Istologia e Medicina Legale, Università degli Studi di Firenze, Viale Morgagni 85, 50134 Firenze, Italy 5 Center of Excellence for Research, Transfer of Research and High Education for the Development of Novel Therapies (DENOthe), Università degli Studi di Firenze, Italy |
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Abstract: | Previous works reported that a mild increase in homocysteine level is a risk factor for cardiovascular and neurodegenerative diseases in humans. Homocysteine thiolactone is a cyclic thioester, most of which is produced by an error-editing function of methionyl-tRNA synthetase, causing in vivo post-translational protein modifications by reacting with the ?-amino group of lysine residues. In cells, the rate of homocysteine thiolactone synthesis is strictly dependent on the levels of the precursor metabolite, homocysteine. In this work, using bovine serum albumin as a model, we investigated the impact of N-homocysteinylation on protein conformation as well as its cellular actions. Previous works demonstrated that protein N-homocysteinylation causes enzyme inactivation, protein aggregation, and precipitation. In addition, in the last few years, several pieces of evidence have indicated that protein unfolding and aggregation are crucial events leading to the formation of amyloid fibrils associated with a wide range of human pathologies. For the first time, our results reveal how the low level of protein N-homocysteinylation can induce mild conformational changes leading to the formation of native-like aggregates evolving over time, producing amyloid-like structures. Taking into account the fact that in humans about 70% of circulating homocysteine is N-linked to blood proteins such as serum albumin and hemoglobin, the results reported in this article could have pathophysiological relevance and could contribute to clarify the mechanisms underlying some pathological consequences described in patients affected by hyperhomocysteinemia. |
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Keywords: | HTL homocysteine thiolactone ANS 1-anilino-8-napthalenesulfonic acid DLS dynamic light scattering BSA bovine serum albumin FBS fetal bovine serum AFM atomic force microscopy TEM transmission electron microscopy ThT thioflavin T MTT 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide PI propidium iodide HSA human serum albumin PBS phosphate-buffered saline |
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