Tetracycline inhibits W7FW14F apomyoglobin fibril extension and keeps the amyloid protein in a prefibrillar, highly cytotoxic state

Abstract

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.

Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity

“…The estimated percentages of secondary structure were: a 10%, b 37%, turn 22%, and unordered 31%. Similarly to what was observed with amyloidogenic proteins, mature fibrils formed by W7FW14F are not cytotoxic, whereas granular aggregates are (Sirangelo et al 2004;Malmo et al 2006). Two factors appear crucial for fibril formation in the case of W7FW14F apomyoglobin: noncovalent interactions and charge effects.…”

“…The cytoxicity of W7FW14F apomyoglobin aggregates was evaluated by the MTT assay. Soluble prefibrillar oligomers have been implicated as the primary toxic species of amyloids; a decrease in reduced MTT levels is generally observed in the presence of these species, whereas fibrils have little or no effect (Booth et al 1997;Kayed et al 2003;Stefani and Dobson 2003;Sirangelo et al 2004;Malmo et al 2006). In particular, we tested the toxicity associated with aggregates formed in the absence and in the presence of heme at the beginning of the aggregation process and after 15 d. The results are shown in Figure 10.…”

“…An accumulation of evidence has led to the hypothesis that the oligomeric precursors mediate cell dysfunction and cell death in degenerative diseases associated with protein misfolding, whereas the fibrillar forms of misfolded proteins are much less toxic or inert (Lambert et al1998;Hartley et al 1999;Bucciantini et al 2002;Lashuel et al 2002a;Stefani and Dobson 2003;Sirangelo et al 2004;Malmo et al 2006). The common pathogenic mechanism, shared by the oligomeric forms of both amyloid diseaseassociated and nonassociated proteins, has been suggested to be intimately related to their similar molecular organization.…”

Effect of Tetracyclines on the Dynamics of Formation and Destructuration of β2-Microglobulin Amyloid Fibrils

“…When doxycycline was added to the ␤2-m solution in the absence of the organic co-solvent but in the presence of phosphate buffer and NaCl, we found very minor changes in the be detected only for the 15 N-1 H correlation of the Phe-22 residue, whereas a number of other backbone amide crosspeaks exhibited distinct and progressive amplitude changes (supplemental Fig. 2).…”

“…So far it has been shown that tetracyclines are able to antagonize the infectivity of the PrP sc aggregates (9), to inhibit in vitro the aggregation of amyloidogenic variants of transthyretin (10), the amyloid-␤ peptide fibrillogenesis (11,12), and amylin fibrillogenesis both in vitro (13) and in vivo (14). The generic anti-aggregation property of tetracyclines has been confirmed in the fibrillogenesis of myoglobin (15), a protein that, although not amyloidogenic in humans, represents a very informative model of the mechanism of conversion of globular proteins into fibrillar assemblies.…”