Tetracycline inhibits W7FW14F apomyoglobin fibril extension and keeps the amyloid protein in a...

©2005 FASEB

The FASEB Journal express article 10.1096/fj.05-4652je. Published online November 29, 2005.

Tetracycline inhibits W7FW14F apomyoglobin fibril extension and keeps the amyloid protein in a prefibrillar, highly cytotoxic state Clorinda Malmo,* Silvia Vilasi,† Clara Iannuzzi,* Silvia Tacchi,† Cesare Cametti,‡ Gaetano Irace,* and Ivana Sirangelo*

*Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, 80138 Napoli; †Dipartimento di Fisica, Università di Perugia, 06100 Perugia; and ‡Dipartimento di Fisica, Università di Roma “La Sapienza,” 00185 Roma, Italy

Corresponding author: Ivana Sirangelo, Dipartimento di Biochimica e Biofisica, Seconda Università di Napoli, Via L. De Crecchio 7, 80138 Napoli, Italy. E-mail: [email protected]

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.

Key words: anti-amyloidogenic compounds ● fibrillogenesis inhibition ● amyloid cytotoxicity

n the protein deposition disorders, a normally soluble protein is deposited in an insoluble form, as either fibrils or amorphous precipitates (1). In addition to systemic amyloidoses, many neurodegenerative disorders, including Alzheimer’s, Huntington’s, Parkinson’s, and

prion diseases, belong to this category. Although the details of the relationship between protein deposition and degeneration are not clear, it is thought that the aggregation process plays an important role in impairing cellular function, ultimately leading to death of involved cells (2, 3). Extensive research is therefore being carried out to tackle the prevention and treatment of these diseases. To achieve this aim, it is crucial to understand the mechanism and molecular details of

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the pathological conversion of amyloidogenic proteins into fibrillar structures. Until very recently, it was thought that only a small number of polypeptide chains associated with clinical disorders were able to form amyloid fibrils. A number of recent studies have, however, shown that proteins unrelated to diseases, under suitable conditions, can form aggregates in vitro with structural and cytotoxic properties that closely resemble those of the amyloid fibrils that are formed in diseased tissues (4–7). These observations have led to the hypothesis that the ability to form amyloid structures is a generic property of proteins resulting from stable interactions primarily involving the main chain that is common to all polypeptides (8–10). Despite major differences in the sequences and structures of the peptides and proteins involved, the fibrillar forms of the aggregates are observed to be similar in their morphologies, typically being unbranched with diameters of 5–10 nm, and to have a common cross-β structure (11–17). These findings suggest that a common molecular mechanism could underlie all such amyloid diseases (10).

The events leading to the transformation of a soluble protein into insoluble amyloid fibrils consist in a conformational change that exposes part of the main chain and hydrophobic residues to the solvent under conditions in which intermolecular interactions can take place. This process leads to the formation of species competent for self-association, which assemble in amyloid fibrils a nucleation-polymerization mechanism (18–21). In most cases, the path of fibril formation begin with prefibrillar kinetic precursors, collectively indicated as protofibrils or soluble oligomeric intermediates, which appear as globules 2.5–5.0 nm in diameter or larger, with an intrinsic tendency to further organize into pore-like annular and tubular structures (22–24). The interest for such prefibrillar intermediates has recently grown, since in most of cases, they have been shown to display the highest cytotoxicity, whereas mature fibrils appear less toxic or even harmless (8, 25–29). The specific mechanism by which these species appear to mediate their toxic effects is not completely understood; probably, toxicity is mediated by common structural features shared by prefibrillar precursors (30, 31). These results have led to the proposal that the molecular basis of cell and tissue impairment may be related to the transient appearance of prefibrillar assemblies, under conditions where their intracellular levels rise due to any dysfunction of the cellular clearing machineries (32).

In our precedent papers (33, 34), we reported that the W7FW14F apomyoglobin mutant is not able to correctly fold at physiological pH and room temperature and forms aggregates that appear granular by electron microscopy, possess extensive β-sheet structure as revealed by circular dichroism, and have the ability to bind specific dyes such as Congo red and thioflavin T (ThT). Mature amyloid fibrils subsequently are generated by populations of prefibrillar aggregates (33). As observed for other amyloidogenic proteins, contrary to mature fibrils, these early aggregates were shown to be toxic to cultured cells when added to the culture media (34).

The identification of molecules that inhibit protein deposition or reverse fibril formation may be a critical step toward a better understanding of the pathophysiology of deposits and their discovery could open new avenues for therapeutic intervention. Numerous compounds have been shown to inhibit specific amyloid fibril formation in vitro (35–42). Two of these agents, i.e., 4′-iodo-4′-deoxydoxorubicin and tetracyclines, have also been reported to reverse amyloid aggregation (38, 40, 41). The present investigation was to test the potential generality of this finding examining the effect of these agents on the formation and dissociation of W7FW14F apomyoglobin amyloid-like fibrils. In particular, we focused our attention on tetracyclines,


which have been reported to inhibit aggregation in several amyloid disorders. This class of antibiotics presents a safe toxicological profile and, thus, is a good candidate for treatment of amyloid-related disease. They bind to amyloid fibrils of human prion protein (PrP), hinder their assembly, and revert the protease resistance of PrP aggregates extracted from brain tissue of patients with Creutzfeldt-Jacob disease (43). In addition, they inhibit transthyretin and Aβ amyloid aggregate formation and disassemble preformed fibrils (38, 41).

Our results demonstrated that tetracycline inhibits W7FW14F apomyoglobin fibrillogenesis but does not cause disaggregation of existing fibrils. Moreover, we investigated the nature and toxicity of the species generated by the action of tetracycline during apomyoglobin amyloid fibrillogenesis. The results showed that the drug produces highly toxic intermediate species, thus suggesting that “amyloid fibril inhibitors” could lead to more pronounced pathology owing to the accumulation of soluble disease-associated oligomers.

MATERIALS AND METHODS

Protein purification

Wild-type and W7FW14F myoglobin were expressed in Escherichia coli M15[pREP4] strain and purified as described previously (33, 34) with some modifications. The wild-type myoglobin was expressed in soluble form and purified under native conditions. The heme was removed by the 2-butanone extraction procedure (44). The W7FW14F mutant was sequestered into insoluble inclusion bodies and purified under denaturing conditions. Refolding was achieved by removing denaturant by dialysis against 10 mM NaH2PO4 pH 2.0, containing decreasing concentrations of urea. The purity of protein samples was assessed by SDS-PAGE. The protein concentration was determined by measuring the absorbance at 280 and 275 nm for the wild-type and mutant protein, respectively, using extinction coefficients of ε280 = 13,500 M−1·cm−1 and ε275 = 3750 M−1·cm−1.

Fibril formation

Solutions of 40 μM W7FW14F apomyoglobin in 10 mM NaH2PO4 pH 2.0 were adjusted at pH 7.0; this results in the formation of protein aggregates in a prefibrillar form that turn into mature fibrils in 7 days (34). The fibril formation was monitored by ThT fluorescence and transmission electron microscopy (TEM).

Incubation of protein aggregates with tetracycline

Tetracycline (Sigma) was dissolved in dimethyl sulfoxide (DMSO) at concentration of 10 mM; typically, a protein/drug molar ratio of 1:5 was used; 40 μM of W7FW14F apomyoglobin in 10 mM NaH2PO4 pH 2.0 were adjusted at pH 7.0 and incubated with 200 μM of tetracycline. Aliquots were taken at different times of incubation.

Atomic force microscopy

Tapping mode atomic force microscopy (AFM) in air was performed by using a Solver Pro scanning probe microscope (NT-MDT, Moscow, Russia). Rectangular silicon cantilevers 100 µm long with resonant frequencies in the range 190–325 kHz and a nominal force constant


between 5.5 and 22.5 N/m (typical 11.5 N/m) were used. AFM images with size between 5 and 15 µm were recorded at a scan rate below 1 Hz with 256 × 256 pixels per image. The cantilevers had integrated tips with a curvature radius of 10 nm and a tip cone angle <220.

The W7FW14F apomyoglobin samples were diluted to a concentration of 50 nM in 2 mM MgCl2. Immediately afterward 10–20 µl of protein solution were deposited onto freshly cleaved ruby mica. The sample was incubated for 2 min, rinsed with water, and blown dry with nitrogen at 0.5 bar.

Dynamic light-scattering measurements

Due to their Brownian motion, particles moving in solution give rise to fluctuations in the intensity of the scattered light. Scattered light at 90° was detected and analyzed with a Brookhaven (BI-9000AT) digital correlator using a laboratory-built setup equipped with a 10 mW He–Ne laser, λ = 632,8 nm. The autocorrelator generates the homodyne intensity–intensity correlation function g(2)(q,t) that, for a Gaussian distribution of the intensity profile of the scattered light, is related to the electric field correlation function g(1)(q,t) by

( ) ( ) ( ) ( )( )212 ,1, tqBgAtqg += (1)

where A and B are the experimental baseline and the constant depending on the optic system, respectively.

For polydisperse particles, g(1)(q,t) is given by

( ) ( ) ( )∫∞

ΓΓ−Γ=0

)1( exp, dtGtqg (2)

Here, G(Γ) is the normalized number distribution function for the decay constant Γ = q2DT, where q = (4πn/λ)sin(θ/2) is the scattering vector with n, the solvent refractive index and DT, the translational diffusion coefficient. The hydrodynamic radius RH is easily calculated from DT through the Stokes–Einstein relationship:

H

BT R

TkDπη6

= (3)

where kB is the Boltzmann’s constant, T the temperature, and η is the viscosity of the medium.

For suspensions having a narrow size distribution, Eq. 2 can be expanded to a second order term, according to (45):

( ) ( )( ) )(2/1,ln 3242

21 tOtqtDqtqg T ++−= μ (4)

where TD is the mean of the diffusion coefficient and μ2 is the variance of the distribution.


For polydisperse systems, particle size distributions were determined from the Laplace inversion of autocorrelation function g(1)(q,t) using the constrained regularization CONTIN algorithm (46), searching for the smoothest nonnegative solution consistent with the experimental data.

Protein solution (40μM) in 10 mM NaH2PO4 pH 4.0 were passed through 0.2 μm cut-off filter before adjustment to pH 7.0. Measurements were performed after 1 and 7 days of tetracycline incubation.

ThT fluorescence measurements

Samples containing 8 μM of protein were mixed with 25 μM ThT in 10 mM phosphate buffer. ThT fluorescence was determined by a Perkin Elmer Life Sciences LS 55 spectrofluorimeter at the excitation and emission wavelengths of 450 and 482 nm, respectively. The fluorescence intensity at 482 nm was corrected by subtracting the emission intensity recorded before the addition of protein to ThT solutions. The fluorescence values of the samples containing only tetracycline were determined and subtracted from the values of the samples containing apomyoglobin fibrils incubated with drug.

Cell culture and incubation with protein aggregates

NIH-3T3 cells (mouse fibroblasts, American Type Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium-high glucose supplemented with 10% bovine calf serum and 3.0 mM glutamine in a 5.0% CO2 humidified environment at 37°C; 50 U/ml penicillin and 50 μg/ml streptomycin were added to the medium. The cells were plated at a density of 100,000 cells/well on 12-well plates in 1 ml of medium for MTT assay and at a density of 30,000 cells on coverslips for DPH staining. After 24 h, protein samples were diluted and added to cell media at final concentration of 20 μM in culture medium. Tetracycline (100μM) was added to cells incubated with the samples treated with the drug. Cells incubated with culture medium without protein in the absence and in the presence of 100 μM tetracycline served as control.

MTT assay

Cell viability was assessed as the inhibition of the ability of cells to reduce the metabolic dye MTT to a blue formazan product (47). After 24 h of incubation with protein samples, cells were rinsed with PBS. One hundred microliters of a stock MTT solution (5 mg/ml in PBS) were then added to 900 μl of DMEM without phenol red containing 10% bovine calf serum/well, and incubation was continued for an additional 3 h. The medium was aspirated, and cells were treated with isopropyl alcohol-0.1 M HCl for 20 min. Levels of reduced MTT were determined by measuring the difference in absorbance at 570 and 690 nm.

DPH staining

After 24 h of incubation with protein samples, slides were fixed by immersing them for 10 min in 4% paraformaldehyde in PBS and stained by adding 10 μM DPH (Molecular Probes) in culture medium for 3 h at 37°C. Cells were rinsed twice in PBS, mounted in antifade, and observed under a Nikon E-1000 fluorescence microscope by using the filter combination UV-2A (330-380 nm excitation, DM 400, BA 420). Images were captured by using a Coolsnal CF (Photometrics) color camera connected with the microscope by using ×10 and ×100 lenses.


RESULTS

The apomyoglobin mutant W7FW14F is known to be not able to correctly fold into a native-like conformation and to form amyloid aggregates at physiological pH (33). The kinetics of in vitro formation of W7FW14F apomyoglobin fibrils shows the classical sigmoidal behavior of aggregating systems, usually attributed to a nucleation-dependent polymerization (48). In addition, the oligomeric intermediates in aggregation (granular aggregates and protofibrils) are the critical toxic species (34). It has been reported that tetracycline exhibits anti-amyloidogenic activity on transthyretin and Aβ amyloid not only by inhibiting the aggregate formation but also by disassembling the preformed fibrils (38, 41). We performed several experiments to determine whether tetracycline shows the same anti-amyloidogenic activity on W7FW14F apomyoglobin mutant.

Tetracycline inhibits W7FW14F apomyoglobin fibrillation

We monitored the effect of tetracycline on fibril formation by using ThT fluorescence assay, AFM, light-scattering, and SDS-PAGE over a period of 15 days. ThT is a fluorescent dye that is known to reliably detect the formation of amyloid fibrils. The emission intensity of the dye is known to increase significantly upon binding to the linear array of β-strands in the amyloid fibrils (49–51). Early apomyoglobin aggregates (formed immediately after pH adjustment to 7.0) 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. This ratio was chosen on the basis of the dose-response effect showed by tetracycline on ThT binding to β 1-42 fibrils (38). Incubation of W7FW14F apomyoglobin aggregates with tetracycline inhibited the ThT fluorescence increase normally observed on time (Fig. 1), thus suggesting that tetracycline inhibits the formation of fibrils from early aggregates. In fact, the amyloidogenic protein incubated with the drug showed a constant low fluorescence level for all observation times, while the control had a logarithmic increase of fluorescence intensity after 3–5 days. Similar results were obtained testing higher protein/drugs molar ratios (1:10 and 1:15) and longer time of tetracycline incubation (21 days).

AFM was used to study the morphological features induced by incubation of W7FW14F apomyoglobin aggregates with tetracycline. 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. 2A–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. 2C–D). The same results were obtained after 15 days of incubation (Fig. 2E–F). These results indicated that the tetracycline influences the fibrillogenesis process of W7FW14F apomyoglobin, inhibiting the formation of mature fibrils from granular aggregates.

Dynamic light-scattering was used to confirm the fibrillogenesis inhibitory properties of tetracycline. The insets of Fig. 3 show the normalized autocorrelation functions g(1)(t) relative to amyloid-forming apomyoglobin incubated in the presence and in the absence of tetracycline. Determinations were performed after 1 and 7 days of incubation at protein/drug molar ratio of 1:5. The size distribution calculated from the normalized autocorrelation function is shown in figure. 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. In particular, after 7 days,


samples incubated with the drug showed two populations of aggregates having average diameters of 0.2 and 1.4 μm. On the other hand, controls exhibited 5 μm average diameter aggregates. Similar results were obtained with a higher protein/drug molar ratio (1:50). These data indicated that tetracycline prevents the conversion of soluble oligomeric intermediates in mature fibrils.

Additional confirmation on fibrillogenesis inhibitory properties of tetracycline was obtained by incubating the early apomyoglobin aggregates with the drug and removing aliquots at different times. These were then centrifuged, and the pellet and supernatant fractions were analyzed by SDS-PAGE. The results are shown in Fig. 4. In the absence of tetracycline the band corresponding to the protein in the pellet increased with time and simultaneously decreased the supernatant fraction indicating the conversion of the soluble oligomeric species in the amyloid fibrils. On the contrary, in the presence of tetracycline the band relative to the supernatant fraction was unchanged.

The effect of tetracycline was also investigated adding the drug to apomyoglobin sample at pH 4.0, i.e., in conditions in which the protein adopts a soluble, monomeric state (33, 34), and, then, neutralized to pH 7.0. The results were similar to those obtained adding the drug to the early apomyoglobin aggregates immediately formed at pH 7.0.

Effect of tetracycline on mature fibrils: fibril disaggregation experiments

We performed experiments to determine whether tetracycline could dissolve preformed fibrils of W7FW14F apomyoglobin. The fibril dissociation was monitored by ThT fluorescence, AFM, and dynamic light scattering. Mature fibrils (7 days aged) of W7FW14F apomyoglobin were incubated with tetracycline at molar ratio of 1:5 and ThT fluorescence was measured immediately and at different times over 15 day period (Fig. 5). The ThT signal remained essentially 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 (15 days) did not determine dissolution of fibrils that were 1 nm wide and 12 μm long, indistinguishable from those of the untreated sample (Fig. 6). 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 into granular aggregates or soluble protein, thus indicating that tetracycline does not show fibril disrupter activity on W7FW14F apomyoglobin fibrils. Dynamic light scattering experiments (data not shown) further confirmed that tetracycline does not have any disruptive activity on apomyoglobin amyloid fibrils.

Effect of tetracycline on cytotoxicity of W7FW14F amyloid aggregates

The nature and toxicity of the species generated by the action of tetracycline was investigated by MTT assay that is a rapid and sensitive indicator of amyloid-mediated toxicity. A decrease in reduced MTT concentrations is generally observed in the presence of soluble prefibrillar oligomers whereas fibrils do not have effect (8, 31, 32, 34). In particular, we tested the toxicity associated with the different species formed during W7FW14F apomyoglobin fibril assembly in the presence and in the absence of drug at a protein/drug molar ratio of 1:5. The results are showed in Fig. 7. As expected, in the absence of tetracycline, early prefibrillar aggregates were toxic (about MTT 55% reduction), whereas mature fibrils (7 and 15 days aged) did not produce


significant decreased level of MTT reduction revealing the absence of toxicity. Incubation with tetracycline for 7 and 15 days produced highly toxic species as compared with untreated samples. These results show that the tetracycline treatment inhibits fibril formation producing soluble oligomeric species with a significant cytotoxicity. Similar results were observed when tetracycline is added to protein sample at pH 4.0 and, then, adjusted at pH 7.0. Incubation with tetracycline at a protein/drug molar ratio of 1:2.5 for 7 and 15 days did not affect cell viability indicating a dose response effect of drug. The effect of higher molar ratio was not tested because tetracycline was toxic to cells.

To further study the effect of tetracycline on fibril growth, we examined cultured fibroblasts incubated with W7FW14F apomyoglobin aggregates formed in the presence and in the absence of the drug, by 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 (52–54). Figure 8 shows the results. In control cells, DPH appears homogeneously distributed on cell membrane, whereas it is not incorporated in the nuclear membrane. Incubation for 24 h with early apomyoglobin aggregates both in the presence and in the absence of tetracycline determines cell aggregation with a concomitant formation of an inhomogeneous DPH coat (Fig. 8B and C). This is probably due to DPH binding to protein aggregates that interact with cell membrane. Figure 8 also shows the effect of protein aggregates grown for 15 days in the absence (D) and in the presence (E) of tetracycline. Cells incubated for 24 h with protein samples grown in the absence of drug did not show aggregation and inhomogeneous fluorescence coating, thus indicating that mature aggregates do not interact with cell membrane. By contrast, protein samples grown in the presence of drug determined the same effect observed for early aggregates.

DISCUSSION

Inhibition of amyloid fibril formation is considered a key prospect therapeutic approach toward amyloid-related diseases. In this respect, molecules that inhibit aggregation may lead to therapies to prevent or control the corresponding diseases. There is growing evidence that early intermediates involved in the aggregation process leading to amyloid structures are much more cytotoxic than the mature amyloid fibrils. The toxicity of these early aggregates seems to derive from an intrinsic ability to impair fundamental cellular processes by interacting with cellular membranes, causing oxidative stress and increase in free Ca2+ that eventually lead to apoptotic or necrotic cell death (30, 55–58). However, there is not evidence that in vivo, in patients, the soluble fibril precursors can have a toxic effect in the absence of amyloid deposits. The finding of molecules that prevent amyloid formation by intercepting the misfolded protein is the common strategy that has been pursued in the development of molecule amyloid inhibitors. Recently, it is been reported that tetracycline exhibits antiamyloidogenic properties by inhibiting aggregate formation and disaggregating preformed fibrils (38, 41).

We tested the effects of tetracycline on the amyloid-forming apomyoglobin mutant. 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 prefibrillar precursors. In fact, using ThT as probe for fibril maturation, we observed that the presence of tetracycline in the incubation medium prevents any further increase of fluorescence intensity. Moreover, AFM images show the lack of mature fibrils and the


persistence of granular aggregates in protein samples incubated with the drug. The inhibition of fibril extension could be caused by the interaction of tetracycline with apomyoglobin oligomers hindering their growth into mature fibrils. Alternatively, tetracycline could interact with monomers still present in solution hampering their recruitment by the already formed oligomers. However, this possibility seems less likely, since no difference was observed adding tetracycline either to the monomeric protein or early aggregates. The persistence of granular aggregates able to interact with cell membranes is responsible for the high cytotoxicity exhibited by W7FW14F mutant apomyoglobin incubated in the presence of tetracycline. Generally, the relatively low cytotoxicity of mature fibrils as compared with precursor aggregates is due to the fact that in the former species most of the regions able to interact with cellular compartments are largely buried and isolated from the cellular environment. In this respect, the increased cytotoxicity of the samples treated with tetracycline could be due to the formation of smaller aggregates that are more reactive because of their higher flexibility that could facilitate the cell membrane interaction.

Using DPH as membrane probe, we observed that the presence of granular aggregates induces cell aggregation; this is probably due to the formation of a membrane-bound protein aggregate coat connecting adjacent cells. Mature fibrils do not cause cell aggregation. Protein samples incubated with tetracycline determine cell aggregation as observed for early soluble oligomeric aggregates.

The persistence of cytotoxicity in protein sample treated with tetracycline is in contrast with previously published results (41). In fact, it has been reported that incubation with tetracycline prevents fibril formation and determines lack of cytotoxicity. The discrepancy could be due to the higher drug concentration used in the cited paper that made necessary to wash the samples to remove the excess of drug before cytotoxic assay. In this manner, if soluble oligomeric prefibrillar aggregates were present, they were removed by the washing treatment. It is worthy to note that several compounds recently discovered to disrupt fibrillar structures prevalently produce oligomeric aggregated species.

Our studies also revealed that tetracycline does not show disrupter activity on W7FW14F apomyoglobin fibrils. In our case, since only few protein segments are involved in the formation of the cross- β-sheet fibrillar structure (unpublished data), the remainder of the molecule could obstacle the accessibility of tetracycline to the fibril core, thus preventing the formation of the preferential noncovalent interactions responsible for the dissolution of the fibrils.

Our findings have important implications for the 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 this respect, a careful usage of tetracycline as fibril inhibitor is indicated because of the accumulation of toxic precursors.

ACKNOWLEDGMENTS

This work has been supported by a grant from Ministero dell’Università e della Ricerca Scientifica e Tecnologica (PRIN 2003).


REFERENCES

1. Carrell, R. W., and Lomas, D. A. (1997) Conformational disease. Lancet 350, 134–138

2. Forno, L. S. (1996) Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. 55, 259–272

3. Thomas, P. J., Qu, B. H., and Pedersen, P. L. (1995) Defective protein folding as a basis of human disease. Trends Biochem. Sci. 20, 456–459

4. Guijarro, J. I., Sunde, M., Jones, J. A., Campbell, I. D., and Dobson, C. M. (1998) Amyloid fibril formation by an SH3 domain. Proc. Natl. Acad. Sci. USA 95, 4224–4228

5. Litvinovich, S. V., Brew, S. A., Aota, S., Akiyama, S. K., Haudenschild, C., and Ingham, K. C. (1998) Formation of amyloid like fibrils by self association of a partially unfolded fibronectin type III module. J. Mol. Biol. 280, 245–258

6. Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ramponi, G., and Dobson, C. M. (1999) Designing conditions for in vitro formation of amyloid protofilaments and fibrils. Proc. Natl. Acad. Sci. USA 96, 3590–3594

7. Fandrich, M., Fletcher, M. A., and Dobson, C. M. (2001) Amyloid fibrils from muscle myoglobin. Nature 410, 165–166

8. Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C. M., and Stefani, M. (2002) Inherent toxicity of aggregates implies a common mechanism for protein misfolding disease. Nature 416, 507–511

9. Dobson, C. M. (2001) The structural basis of protein folding and its links with human disease. Philos. Trans. R. Soc. Lond. B Biol. Sci. 356, 133–145

10. Dobson, C. M. (2003) Protein folding and misfolding. Nature 426, 884–890

11. Serpell, L. C., Sunde, M., and Blake, C. C. (1997) The molecular basis of amyloidosis. Cell. Mol. Life Sci. 53, 871–887

12. Kopito, R. R. (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol. 10, 524–530

13. Jimenez, J. L., Guijarro, J. I., Orlova, E., Zurdo, J., Dobson, C. M., Sunde, M., and Saibil, H. R. (1999) Cryo-electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing. EMBO J. 18, 815–821

14. Serpell, L. C., Sunde, M., Benson, M. D., Tennent, G. A., Pepys, M. B., and Fraser, P. E. (2000) The protofilament substructure of amyloid fibrils. J. Mol. Biol. 300, 1033–1039

15. Makin, O. S., and Serpell, L. C. (2002) Examining the structure of the mature amyloid fibril. Biochem. Soc. Trans. 30, 521–525


16. Hull, R. L., Westermark, G. T., Westermark, P., and Kahn, S. E. (2004) Islet amyloid: a critical entity in the pathogenesis of type 2 diabetes. J. Clin. Endocrinol. Metab. 89, 3629–3643

17. Serpell, L. S. (2000) Alzheimer’s amyloid fibrils: structure and assembly. Biochim. Biophys. Acta 1502, 16–30

18. Booth, D. R., Sunde, M., Bellotti, V., Robinson, C. V., Hutchnson, W. L., Fraser, P. E., Hawkins, P. N., Dobson, C. M., Radford, S. E., Blake, C. C., et al. (1997) Instability, unfolding and aggregation of human lysozyme variants underlying amyloid fibrillogenesis. Nature 385, 787–793

19. Colon, W., and Kelly, J. W. (1992) Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro. Biochemistry 31, 8654–8660

20. Kelly, J. W. (1998) The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. Curr. Opin. Struct. Biol. 8, 101–106

21. Harper, J. D., and Lansbury, P. T., Jr. (1997) Models of amyloid seeding in Alzheimer’s disease and scrapie: mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu. Rev. Biochem. 66, 385–407

22. Lashuel, H. A., Petre, B. M., Wall, J., Simon, M., Nowark, R. J., Waltz, T., and Lansbury, P. T. (2002) α-Synuclein, especially the Parkinson’s disease-associated mutants, forms pore-like annular and tubular protofibrils. J. Mol. Biol. 322, 1089–1102

23. Poirier, M. A., Li, H., Macosko, J., Cail, S., Amzel, M., and Ross, C. A. (2002) Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrillization. J. Biol. Chem. 277, 41,032–41,037

24. Quintas, A., Vaz, D. C., Cardoso, I., Saraiva, M. J., and Broto, R. M. M. (2001) Tetramer dissociation and monomer partial unfolding precedes protofibril formation in amyloidogenic transthyretin variants. J. Biol. Chem. 276, 27,202–27,213

25. Walsh, D. M., and Selkoe, D. J. (2004) Oligomers on the brain: the emerging role of soluble protein aggregates in neurodegeneration. Protein Pept. Lett. 11, 213–228

26. Lambert, M. P., Barlow, A. K., Chromy, B. A., Edwards, C., Freed, R., Liosatos, M., and Morgan, T. E. (1998) Diffusible, nonfibrillar ligands derived from Aβ1–42 are potent central nervous system neurotoxins Proc. Natl. Acad. Sci. USA. 95, 6448−6453.

27. Lashuel, H.A., Hartley, D., Petre, B.M., Waltz, T., and Lansbury, Jr. (2002) Neurogenerative disease: amyloid pores from pathogenic mutations. Nature 418, 291

28. Nilsberth, C., Westlin-Danielsson, A., Eckman, C. B., Condron, M. M., Axelman, K., Forsell, C., Stenh, C., Luthman, J., Teplow, D. B., Younkin, S. G., et al. (2001) The “Arctic” APP mutation (E693G) causes Alzheimer’s disease by enhanced A beta protofibril formation. Nat. Neurosci. 4, 887–893


29. Walsh, D. M., Klyubin, I., Fadeeva, J. V., Cullen, W. K., Anwyl, R., Wolfe, M. S., Rowan, M. J., and Selkoe, D. J. (2002) Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature 416, 535–539

30. Bucciantini, M., Calloni, G., Chiti, F., Formigli, L., Nosi, D., Dobson, C. M., and Stefani, M. (2004) Prefibrillar amyloid protein aggregates share common features of cytotoxicity. J. Biol. Chem. 279, 31,374–31,382

31. Kayed, R., Head, E., Thomson, J. L., Mcintire, T. M., Milton, S. C., Cotman, C. W., and Glabe, C. G. (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300, 486–489

32. Stefani, M., and Dobson, C. M. (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J. Mol. Med. 81, 678–699

33. Sirangelo, I., Malmo, C., Casillo, M., Mezzogiorno, A., Papa, M., and Irace, G. (2002) Tryptophanyl substitution in apomyoglobin determine protein aggregation and amyloid-like fibril formation at physiological pH. J. Biol. Chem. 277, 45,887–45,891

34. Sirangelo, I., Malmo, C., Iannuzzi, C., Mezzogiorno, A., Bianco, M. R., Papa, M., and Irace, G. (2004) Fibrillognesis and cytotoxic activity of the amyloid-forming apomyoglobin mutant W7FW14F. J. Biol. Chem. 279, 13,183–13,189

35. Caughey, B., and Race, R. E. (1992) Potent inhibition of scrapie-associated PrP accumulation by congo red. J. Neurochem. 59, 768–771

36. Merlini, G., Ascari, E., Amboldi, N., Bellotti, V., Arbustivi, E., Perfetti, V., Ferrari, M., Zorzoli, I., Marinane, M. G., and Garini, P. (1995) Interaction of the antjracycline 4’-iodo-4’-deoxydoxorubicin with amyloid fibrils: inhibition of amyloidogenesis. Proc. Natl. Acad. Sci. USA 92, 2959–2963

37. Heiser, V., Scherzinger, E., Boeddrich, A., Nordhoff, E., Lurz, R., Schugardt, N., Lehrach, H., and Wanker, E. E. (2000) Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: implications for Huntington’s disease therapy. Proc. Natl. Acad. Sci. USA 97, 6739–6744

38. Forloni, G., Colombo, L., Girola, L., Tagliavini, F., and Salmona, M. (2001) Anti-amyloidogenic activity of tetracyclines: studies in vitro. FEBS Lett. 487, 404–407

39. Howlett, D. R., Perry, A. E., Godfrey, F., Swatton, J. E., Jennings, K. H., Spitzfaden, C., Wadsworth, H., Wood, S. J., and Markwell, R. E. (1999) Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans. Biochem. J. 340, 283–289

40. Sebastiano, M. P., Merlini, G., Saraiva, M. J., and Damas, A. M. (2000) The molecular interaction of 4′-iodo-4′-deoxydoxorubicin with Leu-55Pro transthyretin “amyloid-like” oligomer leading to disaggregation. Biochem. J. 351, 273–279


41. Cardoso, I., Merlini, G., and Saraiva, M. J. (2003) 4′-iodo-4′-deoxydoxorubicin and tetracyclines disrupt transthyretin amyloid fibrils in vitro producine noncytotoxic species: screening for TTR fibril disrupters. FASEB J. 17, 803–809

42. Li, J., Zhu, M., Manning-Bog, A. B., Di Monte, D. A., and Fink, A. L. (2004) Dopamine and L-dopa disaggregate amyloid fibrils: implication for Parkinson’s and Alzheimer’s disease. FASEB J. 18, 962–964

43. Tagliavini, F., Forloni, G., Colombo, L., Rossi, G., Girola, L., Canciani, B., Angeretti, N., Giampaolo, L., Peressini, E., Awan, T., et al. (2000) Tetracicline affects abnormal properties of synthetic PrP peptides and PrPSc in vitro. J. Mol. Biol. 300, 1309–1322

44. Teale, F. W. J. (1959) Cleavage of the heme protein by acid methylethylketone. Biochim. Biophys. Acta 35, 543

45. Koppel, D. E. (1972) Analysis of macromolecular polydispersity in intensity correlation spettroscopy:the method of cumulants. J. Chem. Phys. 57, 4814–4820

46. Provencher, S. W. (1982) CONTIN: a general purpose constrained regularization program for inverting noisy linear algebraic and integral equations. Comp. Phys. Commun. 27, 229–242

47. Hansen, M. B., Nielsen, S. E., and Berg, K. (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods 119, 203–210

48. Jarret, J. T., and Lansbury, P. T., Jr. (1992) Amyloid fibril formation requires a chemically discriminating nucleation event: studies of an amyloidogenic sequence from the bacterial protein OsmB. Biochemistry 31, 12,345–12,352

49. Naiki, H., Higuchi, K., Hosowaka, M., and Takeda, T. (1989) Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1. Anal. Biochem. 177, 244–249

50. Naiki, H., Higuchi, K., Nakakuki, K., and Takeda, T. (1991) Kinetic analysis of amyloid fibril polymerization in vitro. Lab. Invest. 65, 104–110

51. LeVine, H., III (1993) Thioflavine T interaction with synthetic Alzheimer’s disease beta-amyloid peptides: detection of amyloid aggregation in solution. Protein Sci. 2, 404–410

52. Lentz, B. R., Barenholz, Y., and Thompson, T. E. (1976) Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 1. Single component phosphatidylcholine liposomes. Biochemistry 15, 4521–4528

53. Lentz, B. R., Barenholz, Y., and Thompson, T. E. (1976) Fluorescence depolarization studies of phase transitions and fluidity in phospholipid bilayers. 2 Two-component phosphatidylcholine liposomes. Biochemistry 15, 4529–4537


54. Chen, L. A., Dale, R. E., Roth, S., and Brand, L. (1977) Nanosecond time-dependent fluorescence depolarization of diphenylhexatriene in dimyristoyllecithin vesicles and the determination of “microviscosity.” J. Biol. Chem. 252, 2163–2169

55. Lin, H., Bhatia, R., and Lal, R. (2001) Amyloid β protein forms ion channels: implications for Alzheimer’s diseases pathophysiology. FASEB J. 15, 2433–2444

56. Hirakura, Y., Carreras, I., Sipe, J. D., and Kagan, B. L. (2002) Channel formation by serum amyloid A: a potential mechanism for amyloid pathogenesis and host defence. Amyloid 9, 13–23

57. Mirzabekov, T. A., Lin, M. C., and Kagan, B. L. (1996) Pore formation by the cytotoxic islet amyloid peptide amylin. J. Biol. Chem. 271, 1988–1992

58. Lin, M. C., Mirzabekov, T. A., and Kagan, B. L. (1997) Channel formation by a neurotoxic prion protein fragment. J. Biol. Chem. 272, 44–47

Received June 26, 2005; accepted October 10, 2005.


Fig. 1

Figure 1. W7FW14F apomyoglobin fibrillogenesis monitored by ThT fluorescence emission in the presence (�) and in the absence (♦) of tetracycline. Protein concentration was 8 µM, and protein/drug molar ratio was 1:5. The presence of tetracycline inhibits the ThT fluorescence increase normally observed on time.


Fig. 2

Figure 2. W7FW14F apomyoglobin fibrillogenesis monitored by AFM in the presence (right panels) and in the absence (left panels) of tetracycline. Scale on right represents height of pixels in image; lighter color represents 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 protein in an oligomeric granular state.


Fig. 3

Figure 3. Dynamic light scattering of W7FW14F apomyoglobin in the presence (right panels) and in the absence (left panels) of tetracycline. Aliquots of protein were taken at 1 (A, B) and 7 days (C, D) from the beginning of fibrillogenesis process. Figures show size distribution of scattering particles calculated from the autocorrelation function (shown in insets). It is evident that incubation with tetracycline decreases average diameter of scattering particles.


Fig. 4

Figure 4. Effect of tetracycline on W7FW14F apomyoglobin aggregation analyzed by SDS-PAGE. Aliquots of protein were taken at 0, 7, and 15 days from beginning of fibrillogenesis process and centrifuged at 20,000 g for 15 min. Lanes A and C: pellet; lanes B and D: supernatant. Supernatant fraction remains unchanged in protein samples incubated with tetracycline.


Fig. 5

Figure 5. Effect of tetracycline on preformed W7FW14F apomyoglobin fibrils monitored by ThT fluorescence. Fibrils of 7 days aged were incubated in the absence (♦) and in the presence (�) of tetracycline. Protein concentration was 8 µM, and protein/drug molar ratio was 1:5. Amount of fibrils did not decrease in the presence of tetracycline during incubation time.


Fig. 6

Figure 6. AFM images of preformed fibrils incubated in the absence (A) and in the presence (B) of tetracycline for 15 days. Scale on right represents height of pixels in image. The presence of drug did not disrupt fibril formations.


Fig. 7

Figure 7. 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 beginning of fibrillogenesis process and incubated for 24 h with cells. Data are expressed as average percentage 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, and tetracycline concentration was 100 µM. Only early soluble oligomeric aggregates caused a significant decreases in reduced MTT levels (P<0.01).


Fig. 8

Figure 8. DPH staining 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). Aliquots of protein were taken at 0 (B, C) and 15 days (D, E) from beginning of fibrillogenesis process and incubated for 24 h with cells. A) Cell control. Protein concentration was 20 µM, and tetracycline concentration was 100 µM. Early soluble aggregates and tetracycline-treated samples determined cell aggregation. Mature fibrils did not induce cell aggregation.


Tetracycline inhibits W7FW14F apomyoglobin fibril extension and keeps the amyloid protein in a...

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