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The FASEB Journal FJ Express
Tetracycline inhibits W7FW14F apomyoglobin fibril
extension and keeps the amyloid protein in
a pre-fibrillar, highly cytotoxic state

Clorinda Malmo,* Silvia Vilasi,† Clara Iannuzzi,* Silvia Tacchi,† Cesare Cametti‡,
Gaetano Irace,* and Ivana Sirangelo*,1
*Dipartimento di Biochimica e Biofisica, Seconda Universita` di Napoli, Naples, Italy; †Dipartimento
di Fisica, Universita` di Perugia, Perugia, Italy; and ‡Dipartimento di Fisica, Universita` di Roma “La
Sapienza, Rome, Italy
To read the full text of this article, go to;
doi: 10.1096/fj.05-4652fje

average diameter of the aggregates formed in thepresence of tetracycline was lower than that of con- Our aim was to test the general potentiality of tetracy- trols both at shorter and longer time of incubation.
cline as antiamyloidogenic agent probing its effect on Finally, SDS-PAGE analysis of pellet and supernatant the amyloid forming apomyoglobin mutant W7FW14F.
fractions of W7FW14F apomyoglobin aggregatesshowed that the protein concentration in the pelletdoes not change in time in samples incubated with PRINCIPAL FINDINGS
1. Tetracycline inhibits W7FW14F
2. Mature fibrils are not disaggregated by tetracycline
apomyoglobin fibrillogenesis
Fibril dissociation was monitored by ThT fluorescence, We monitored the effect of tetracycline on fibril AFM, and dynamic light scattering. Mature fibrils of formation by using thioflavin T (ThT) fluorescence W7FW14F apomyoglobin were incubated with tetracy- assay, atomic force microscopy (AFM), and light- cline at molar ratio of 1:5 and ThT fluorescence was scattering. Early apomyoglobin aggregates were incu- followed in time. The ThT signal remained constant, bated with tetracycline at a protein/drug molar ratio thus indicating that tetracycline does not dissolve apo- of 1:5 and aliquots of sample were tested for ThT myoglobin fibrils. These data were confirmed by AFM fluorescence assay at different times. Tetracycline images showing that fibrils are present after 8 days of inhibited the ThT fluorescence increase normally tetracycline incubation. Even longer incubation times observed on time. The morphological features in- did not determine dissolution of fibrils. Further addi- duced by incubation of W7FW14F apomyoglobin tions of drug to preparation during incubation time aggregates with tetracycline were examined by AFM.
(1:10, 1:15, and 1:25 protein/drug molar ratio) did not Only globular structures (5 nm radius) were ob- lead to disruption of the fibrillar material. Dynamic served in the early stages of aggregation both in the light scattering experiments further confirmed that presence and in the absence of tetracycline (Fig.
tetracycline does not have any disruptive activity on 1A–B). After 7 days from the beginning of the
aggregation process, the globular structures disap-peared almost completely and mature fibrils were 3. The species produced by tetracycline action are
formed in the protein control, while in the presence highly cytotoxic
of tetracycline only globular structures were observed
(Fig. 1C–D). The same results were obtained after 15
We tested the toxicity associated with the different days of incubation (Fig. 1E–F). These results indi- species formed during W7FW14F apomyoglobin fibril cated that the tetracycline inhibits the fibrillogenesis assembly in the presence and in the absence of drug by process of W7FW14F apomyoglobin preventing the MTT assay (Fig. 2). Early pre-fibrillar aggregates were
formation of mature fibrils from early granular ag-gregates. Dynamic light-scattering was used to con-firm the fibrillogenesis inhibitory properties of tetra- 1 Corresponding author: Dipartimento di Biochimica e cycline. The size distribution calculated from the Biofisica, Seconda Universita` di Napoli, Via L. De Crecchio 7, normalized autocorrelation function showed that the Napoli 80138, Italy. E-mail: Figure 2. Cell viability of NIH-3T3 cells exposed to W7FW14F
apomyoglobin aggregates formed in the presence and in the
absence of tetracycline (protein/drug molar ratio was 1:5)
detected by MTT assay. Aliquots of protein were taken at 0 (a,
b), 7 (c, d), and 15 days (e, f) from the beginning of fibrillo-
genesis process and incubated for 24 h with cells. Data are
expressed as average percent of MTT reduction Ϯsd relative to
cells treated with medium alone or medium plus tetracycline,
from triplicate wells from 5 separate experiments. Protein con-
centration was 20 ␮M, tetracycline concentration was 100 ␮M.
Only early soluble oligomeric aggregates caused a significant
decreases in reduced MTT levels (PϽ0.01).
Figure 1. W7FW14F apomyoglobin fibrillogenesis monitored
by AFM in the presence (right panels) and in the absence
(left panels) of tetracycline. The scale on the right represents
Our data indicate that the presence of tetracycline inter- the height of pixels in the image; the lighter color represents feres with W7FW14F apomyoglobin fibril extension rather the higher height from mica surface. Aliquots of protein were than to inhibit initiation of fibrillogenesis, keeping the taken at 0 (A, B), 7 (C, D), and 15 days (E, F) from the protein in the status of highly cytotoxic pre-fibrillar pre- beginning of fibrillogenesis process. Protein/drug molar cursors (Fig. 3). Our studies also revealed that tetracycline
ratio was 1:5. The presence of tetracycline inhibits fibril does not show disrupter activity on W7FW14F apomyoglo- formation and keeps the protein in an oligomeric granularstate.
bin mature fibrils. These results are in contrast withprevious published data indicating that tetracycline deter-mines lack of toxicity and fibril dissociation. In thisrespect, our findings have important implications for toxic (about MTT 55% reduction), whereas mature development of therapeutic agents of protein deposition fibrils (aged 7 and 15 days) did not. Incubation with diseases. If fibrillar proteins contribute to cell degenera- tetracycline for 7 and 15 days produced highly toxic tion, then the disassembly of fibrils may reverse or slow species as compared with untreated samples. These down disease progression; however, if the action of ther- results show that the tetracycline-induced inhibition of apeutic agents produces intermediates of fibrillation fibril formation produces oligomeric species with a and/or products of fibril disaggregation, then their accu- mulation could be harmful. In conclusion, a careful usage Finally, we examined the effect of W7FW14F apo- of tetracycline as fibril inhibitor is indicated because of myoglobin aggregates formed in the absence and in the accumulation of toxic precursors.
the presence of tetracycline on cultured fibroblastsusing 1,6-diphenyl-1,3,5-hexatriene (DPH) staining.
DPH is a rod-shaped molecule soluble and highlyfluorescent only in a hydrophobic environment, likethat existing in the interior of cell membranes.
Incubation for 24 h with early apomyoglobin aggre-gates determines cell aggregation. Cells incubatedfor 24 h with mature fibrils did not show aggregation,thus indicating that they do not interact with cellmembrane. By contrast, protein samples grown in thepresence of drug determined the same effect ob- Figure 3. Schematic diagram. Tetracycline inhibits fibril ex-
tension and keeps the protein in a highly cytotoxic state.


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