HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 20/2/346 05-4652fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Malmo, C. Articles by Sirangelo, I. Search for Related Content PubMed PubMed Citation Articles by Malmo, C. Articles by Sirangelo, I.

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 Università di Napoli, Naples, Italy;
Dipartimento di Fisica, Università di Perugia, Perugia, Italy; and
Dipartimento di Fisica, Università di Roma "La Sapienza, Rome, Italy

¹ Corresponding author: Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Via L. De Crecchio 7, Napoli 80138, Italy. E-mail: ivana.sirangelo{at}unina2.it

SPECIFIC AIMS

Our aim was to test the general potentiality of tetracycline as antiamyloidogenic agent probing its effect on the amyloid forming apomyoglobin mutant W7FW14F.

PRINCIPAL FINDINGS

1. Tetracycline inhibits W7FW14F apomyoglobin fibrillogenesis
We monitored the effect of tetracycline on fibril formation by using thioflavin T (ThT) fluorescence assay, atomic force microscopy (AFM), and light-scattering. Early apomyoglobin aggregates were incubated with tetracycline at a protein/drug molar ratio of 1:5 and aliquots of sample were tested for ThT fluorescence assay at different times. Tetracycline inhibited the ThT fluorescence increase normally observed on time. The morphological features induced by incubation of W7FW14F apomyoglobin aggregates with tetracycline were examined by AFM. Only globular structures (5 nm radius) were observed in the early stages of aggregation both in the presence and in the absence of tetracycline (Fig. 1 A–B). After 7 days from the beginning of the aggregation process, the globular structures disappeared almost completely and mature fibrils were formed in the protein control, while in the presence of tetracycline only globular structures were observed (Fig. 1C-D ). The same results were obtained after 15 days of incubation (Fig. 1E-F ). These results indicated that the tetracycline inhibits the fibrillogenesis process of W7FW14F apomyoglobin preventing the formation of mature fibrils from early granular aggregates. Dynamic light-scattering was used to confirm the fibrillogenesis inhibitory properties of tetracycline. The size distribution calculated from the normalized autocorrelation function showed that the average diameter of the aggregates formed in the presence of tetracycline was lower than that of controls both at shorter and longer time of incubation. Finally, SDS-PAGE analysis of pellet and supernatant fractions of W7FW14F apomyoglobin aggregates showed that the protein concentration in the pellet does not change in time in samples incubated with tetracycline.

View larger version (102K): [in this window] [in a new window]   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 the height of pixels in the image; the lighter color represents the higher height from mica surface. Aliquots of protein were taken at 0 (A, B), 7 (C, D), and 15 days (E, F) from the beginning of fibrillogenesis process. Protein/drug molar ratio was 1:5. The presence of tetracycline inhibits fibril formation and keeps the protein in an oligomeric granular state.

2. Mature fibrils are not disaggregated by tetracycline
Fibril dissociation was monitored by ThT fluorescence, AFM, and dynamic light scattering. Mature fibrils of W7FW14F apomyoglobin were incubated with tetracycline at molar ratio of 1:5 and ThT fluorescence was followed in time. The ThT signal remained constant, thus indicating that tetracycline does not dissolve apomyoglobin fibrils. These data were confirmed by AFM images showing that fibrils are present after 8 days of tetracycline incubation. Even longer incubation times did not determine dissolution of fibrils. Further additions of drug to preparation during incubation time (1:10, 1:15, and 1:25 protein/drug molar ratio) did not lead to disruption of the fibrillar material. Dynamic light scattering experiments further confirmed that tetracycline does not have any disruptive activity on apomyoglobin amyloid fibrils.

3. The species produced by tetracycline action are highly cytotoxic
We tested the toxicity associated with the different species formed during W7FW14F apomyoglobin fibril assembly in the presence and in the absence of drug by MTT assay (Fig. 2 ). Early pre-fibrillar aggregates were toxic (about MTT 55% reduction), whereas mature fibrils (aged 7 and 15 days) did not. Incubation with tetracycline for 7 and 15 days produced highly toxic species as compared with untreated samples. These results show that the tetracycline-induced inhibition of fibril formation produces oligomeric species with a significantly high cytotoxicity.

View larger version (20K): [in this window] [in a new window]   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 fibrillogenesis process and incubated for 24 h with cells. Data are expressed as average percent of MTT reduction ±SD relative to cells treated with medium alone or medium plus tetracycline, from triplicate wells from 5 separate experiments. Protein concentration was 20 µM, tetracycline concentration was 100 µM. Only early soluble oligomeric aggregates caused a significant decreases in reduced MTT levels (P<0.01).

Finally, we examined the effect of W7FW14F apomyoglobin aggregates formed in the absence and in the presence of tetracycline on cultured fibroblasts using 1,6-diphenyl-1,3,5-hexatriene (DPH) staining. DPH is a rod-shaped molecule soluble and highly fluorescent only in a hydrophobic environment, like that existing in the interior of cell membranes. Incubation for 24 h with early apomyoglobin aggregates determines cell aggregation. Cells incubated for 24 h with mature fibrils did not show aggregation, thus indicating that they do not interact with cell membrane. By contrast, protein samples grown in the presence of drug determined the same effect observed for early aggregates.

CONCLUSIONS AND SIGNIFICANCE

Our data indicate that the presence of tetracycline interferes with W7FW14F apomyoglobin fibril extension rather than to inhibit initiation of fibrillogenesis, keeping the protein in the status of highly cytotoxic pre-fibrillar precursors (Fig. 3 ). Our studies also revealed that tetracycline does not show disrupter activity on W7FW14F apomyoglobin mature fibrils. These results are in contrast with previous published data indicating that tetracycline determines lack of toxicity and fibril dissociation. In this respect, our findings have important implications for development of therapeutic agents of protein deposition diseases. If fibrillar proteins contribute to cell degeneration, then the disassembly of fibrils may reverse or slow down disease progression; however, if the action of therapeutic agents produces intermediates of fibrillation and/or products of fibril disaggregation, then their accumulation could be harmful. In conclusion, a careful usage of tetracycline as fibril inhibitor is indicated because of the accumulation of toxic precursors.

View larger version (11K): [in this window] [in a new window]   Figure 3. Schematic diagram. Tetracycline inhibits fibril extension and keeps the protein in a highly cytotoxic state.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4652fje;

This article has been cited by other articles:

				C. Iannuzzi, S. Vilasi, M. Portaccio, G. Irace, and I. Sirangelo Heme binding inhibits the fibrillization of amyloidogenic apomyoglobin and determines lack of aggregate cytotoxicity Protein Sci., March 1, 2007; 16(3): 507 - 516. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 20/2/346 05-4652fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Malmo, C. Articles by Sirangelo, I. Search for Related Content PubMed PubMed Citation Articles by Malmo, C. Articles by Sirangelo, I.