Supramolecular amyloid-like assembly of the polypeptide sequence coded by exon 30 of human tropoelastin.

Elastin is known to self-aggregate in twisted-rope filaments. However, an ultrastructural organization different from the fibrils typical of elastin, but rather similar to those shown by amyloid networks, is shown by the polypeptide sequence encoded by exon 30 of human tropoelastin. To better understand the molecular properties of this sequence to give amyloid fibers, we used CD, NMR, and FTIR (Fourier transform infrared spectroscopy) to identify the structural characteristics of the peptide. In this study, we have demonstrated, by FTIR, that antiparallel beta-sheet conformation is predominant in the exon 30 fibers. These physical-chemical studies were combined with transmission electron microscopy and atomic force microscopy to analyze the supramolecular structure of the self-assembled aggregate. These studies show the presence of fibrils that interact side-by-side probably originating from an extensive self-interaction of elemental cross beta-structures. Similar sequences, of the general type XGGZG(X, Z = V, L, A, I), are widely found in many proteins such as collagens IV and XVII, major prion protein precursor, amyloid beta A4 precursor protein-binding family, etc., thus suggesting that this sequence could be involved in contributing to the self-assembly of amyloid fibers even in other proteins.

Elastin is the protein responsible for the elasticity of skin, arteries, and lung whose elastic properties and resilience depend on its supramolecular organization and cross-linking into fibers and lamellae (1)(2)(3)(4). Although, the ability of the elastin precursor molecule to undergo self-aggregation is an important property of the assembly process, the steps involved in elastic fiber formation are not precisely known (5). Actually, it has been widely demonstrated that even short peptides and simple polymeric sequences belonging to elastin are able to give filaments with twisted-rope, hierarchical supramolecular organization similar to that found for the entire protein (6,7). Interestingly, Miao et al. (8) have found that some mutated polypeptide sequences encoded by exons of human tropoelastin exhibit an ultrastructural organization different from the fibrils typical of elastin but rather similar to those found for amyloid networks, constituted by helical fibrils several microns long and about 10 nm wide (9).
Recently, Kozel et al. (10) have demonstrated that a polypeptide consisting of the sequence coded by exon 30 of bovine tropoelastin formed amyloid-like fibrils.
The fibrous structures referred to as amyloids are widely found (11)(12)(13) and are responsible for various medical disorders, e.g. bovine spongiform encephalopathies or Creuzfeld-Jacob disease, Alzheimer disease, type II diabetes, or Huntington disease (14).
From a structural point of view, amyloid fibrils contain significant amounts of the cross-␤ structural motif originally revealed by x-ray diffraction (9). According to current opinion (see, however, Ref. 15), the cross-␤-structure originates antiparallel ␤-sheets extending over the length of the fibril, formed by ␤-strands that run nearly perpendicular to the long axis of the fibril. Interestingly, ␤-strands, whose role in elastin assembly has to be ascertained, have been detected in elastin in the solid state (16 -18).
Recently, we found that the sequence encoded by exon 30 (EX30) 1 of human tropoelastin is the only sequence in the molecule whose CD spectra roughly resemble a ␤-sheet conformation (19) and that the conformation of EX30 peptide is temperature-and concentration-dependent (20).
Currently, the criteria to be adopted to assess the presence of amyloid fibrils are quite controversial. On summarizing, positive results for Congo Red binding together with the presence of ␤-sheet secondary structure revealed by FTIR and/or CD and NMR spectroscopy could be considered sufficient evidences for the presence of amyloid fibers (21). Accordingly, to better understand the molecular properties of EX30, we used CD, NMR, and FTIR to identify the structural characteristics of the peptide. These physical-chemical studies were combined with transmission electron microscopy and atomic force microscopy to analyze the supramolecular structure of the self-assembled aggregate. Our results suggest that the peculiar propensity of EX30 peptide to adopt a ␤-sheet conformation is the crucial element that favors the adoption of amyloid-like organization.

MATERIALS AND METHODS
CD Spectroscopy-CD spectra for the peptides were obtained using a Jasco J-600 Spectropolarimeter at various temperatures and at concentrations of 0.1 and 1.0 mg/ml in water by using cells of 0.1 and 0.01 cm, respectively. Spectra were acquired in the range 190 -250 nm at a temperature of 25°C by taking points every 0.1 nm, with 20 nm min Ϫ1 scan rate, an integration time of 2 s, and a 1 nm bandwidth. The data are expressed in terms of [⌰], the molar ellipticity in units of degree cm 2 dmol Ϫ1 .
Nuclear Magnetic Resonance Spectroscopy-All 1 H NMR experiments were performed on a Varian Unity INOVA 500 MHz spectrometer equipped with a 5-mm triple-resonance probe and z-axial gradi-ents. The purified peptides were dissolved in 700 l of H 2 O/D 2 O (90/10) or TFE-d 3 /H 2 O (80/20), containing 0.1 mM 3-(trimethylsily1)-1-propane sulfonic acid as internal reference standard at 0 ppm. Usually 1.5 mM peptide solutions were used. The 1 H NMR spectra were acquired at 15°, 25°and 45°C in TFE and also at 15 and 25°C in water. One-dimensional spectra were acquired in Fourier mode with quadrature detection, and the water signal was suppressed by a 2.5-s presaturation pulse. Two-dimensional TOCSY (22) and NOESY (23) spectra were collected in the phase-sensitive mode using the States method (24). Typical data were 2048 complex data points, 8 or 32 transients and 256 increments. Relaxation delays were set to 2.5 s, and spinlock (MLEV-17) mixing time was 80 ms for TOCSY, while 150 -250 ms mixing time was applied to NOESY experiments. Shifted sine bell squared weighting and zero filling to 2048 ϫ 2048 was applied before Fourier transformation. The residual HDO signal was suppressed by presaturation for TOCSY, while WATERGATE pulse sequence was used for NOESY experiments (25). Amide proton temperature coefficients were usually measured from one-dimensional 1 H NMR spectra recorded in 5°C increments from 20 to 45°C; for overlapping resonances a series of TOCSY were recorded to measure the amide temperature coefficients. Spectra were processed and analyzed by VNMR Version 6.1C software (Varian, Palo Alto, CA).
Resonance Assignments of Elastin Peptide-The EX30 peptide was analyzed by 1 H NMR spectroscopy in water (H 2 O/D 2 O 90/10) and mixed aqueous/organic solution (TFE-d 3 /H 2 O 80/20) at 25°C. The resonance assignment of the 1 H NMR spectra was made by standard sequential assignment procedures (26) and accomplished by a combined analysis of two-dimensinoal TOCSY and two-dimensional NOESY spectra. TOCSY spectra were used to identify spin systems, while NOESY spectra permitted the assignment of resonance to individual amino acids through sequential NOE connectivities. The presence of a considerable number of glycine residues in the peptide sequence (52%) made the assignment of the resonance to the different residues in the sequence difficult. Nevertheless, in TFE-d 3 /H 2 O solution the complete chemical shift assignments and NOE analysis of the peptide were accomplished. In H 2 O/D 2 O a stronger degeneracy of the signals precluded the complete NOE analysis of the glycine residues resonance.
The presence of a proline in the EX30 peptide sequence allows cis/trans isomerization to occur in both solution conditions. Nevertheless, we identified only one major conformation (Ͼ90%) with all-trans proline conformers. Thus the conformational analysis has been carried out only for the major all-trans peptide molecules.
Congo Red Binding-1.0 ml of a 2 mM solution of the EX30 peptide in Tris (50 mM), NaCl (1.5 M), and CaCl 2 (1.0 mM) (pH 7.0) solution was incubated overnight at room temperature. The solution was stirred to suspend the precipitated peptide, and an absorbance spectrum from 400 to 600 nm was collected with a Cary UV spectrophotometer using quartz cuvettes. Congo Red was added to a final concentration of 3.0 M, and the spectrum was recorded.
A third spectrum of the unbound dye was also collected. The spectrum of peptide alone was subtracted from the spectrum of Congo Red with peptide to correct for the turbidity of the sample due to the precipitated material.
FTIR Spectroscopy-EX30 peptide was analyzed by FTIR either as a lyophilized powder or as precipitated fibers in KBr pellets. The spectra were recorded on a Jasco spectrometer using a resolution of 4 cm Ϫ1 and then smoothed by using the Savintky-Goolay algorithm. The samples were examined at the solid state in KBr pellets (1 mg/100 mg). The decomposition of FTIR spectra was obtained using GRAMS32 software. The percentage of Gaussian and Lorentzian functions was fixed at the 8:2 ratio.
Electron Microscopy-EX30 peptide (1 mg/ml) was solubilized in Tyrode's physiological solution pH 7.2 at room temperature and incubated at 20, 35, and 50°C for times from 1 up to 48 h. At fixed intervals, 10 l of the solution were (a) placed on Formvar and carbon-coated copper grids, negatively stained with a few drops of 1% uranyl acetate in bidistilled water, air-dried, and observed by transmission electron microscopy and (b) spread on freshly cleaved mica, rotary-shadowed with platinum-carbon from an angle of 11 degrees, and covered by evaporated carbon. The replica was floated on bidistilled water, collected on copper grids, air-dried, and observed under the electron microscope.
Atomic Force Microscopy-About 10 l of the peptide solutions were spread on freshly cleaved mica as described above and salts removed by washing the specimens with twice-distilled water. Samples were stored sealed in a Petri dish until observed by the scanning force microscope (Park Autoprobe). Specimens were observed at room temperature and by the tapping mode.

RESULTS
Sequence Analysis-The sequence of EX30 peptide is GLV-GAAGLGGLGVGGLGVPGVGGLG. In the alternate succession of hydrophobic and cross-linking domains, peculiar of tropoelastin (27), this peptide belongs to the C-terminal hydrophobic domains of human tropoelastin and has been indicated as fundamental for a correct assembly of elastin (10). It is a glycine-rich domain, containing 13 glycine residues up to 25 residues, and contains only hydrophobic residues. A certain homology is evident among sequences of human, bovine, porcine, and ovine tropoelastin (Table I), while in other mammalian, such as in mouse, the EX30-coded domain is constituted by a nine time-repeated pentamer LGAGG. At variance with other hydrophobic domains of human tropoelastin it has no tendency to coacervate (28) but forms peptide aggregates with fibrillar structure instead. Similar sequences, of the general type XGGZG (X, Z ϭ V, L, A, I), are widely found in many proteins such as collagens IV and XVII, fibrillin 2 precursor, flagelliform silk protein, major ampullate gland dragline silk protein, major prion protein precursor, lamprin precursor, and in amyloid ␤ A4 precursor protein-binding family, etc.
CD Difference Spectra-CD spectroscopy is a useful tool in identifying the global conformation of a peptide or a protein. It is also helpful in highlighting the conformational changes as a function of environmental changes, such as temperature, solvent, salt, and peptide concentration. The CD spectra of a protein can be assumed to be the linear combination of the spectra of the secondary structural elements, so difference spectra of CD spectra recorded in different conditions should point out the conformational changes involved. CD spectra of EX30 peptide were recorded in different solvents and at different temperatures.
The CD difference spectra of EX30 in water are shown in Fig. 1. The curve obtained by subtracting the spectra at the two extreme temperatures, 70 and 0°C, shows a maximum at 202 nm and a minimum at 217 nm. This type of curve is typical of a mixture of ␤-sheet and unordered conformations and indicates the dominance of the ␤ conformation on increasing the temperatures. At lower temperatures the CD spectra clearly indicate the dominance of PPII conformation (20). A similar effect can be obtained by increasing the peptide concentration. In fact, the CD difference spectrum, obtained by subtracting the curve at 0.1 mg/ml to that at 1.0 mg/ml, shows a maximum at 195 nm and a small minimum at 205 nm (Fig. 2). These spectral features can most likely be attributed to a distorted ␤-sheet conformation that gives rise to a spectral blue shift and to a strong decrease in the negative band relative to the standard CD of the ␤-structure. The decrease in the negative band is suggested to be due to a shift of the dihedral that adopts higher values thereby rendering it markedly less negative (see Fig. 13 in Ref. 29).
NMR Spectroscopy-The EX30 peptide has been analyzed in H 2 O/D 2 O 90/10 and in less polar solvent conditions (TFE-d 3 / H 2 O 70/30), which better mimic the highly hydrophobic microenvironment of the peptide sequences when inserted in the whole protein.
Several NMR parameters were used to deduce the secondary Ovine * * *:**** ***** *** :* structure of EX30 peptide: chemical shift index, temperature coefficients of amide chemical shifts, 3 J (HN-H␣) coupling constants and the pattern of intra-and inter-residue NOEs. Chemical shift index analysis (30) is based on the observation that some secondary structures present chemical shifts for some atoms (H␣, C␣, CЈ), which deviate significantly, from random coil values. In particular, in 1 H NMR spectroscopy, chemical shift index is a valuable tool for identifying ␣-helix and ␤-sheet simply by comparing the chemical shifts of H␣ protons of the residues with the tabulated random coil values. The H␣ protons of the EX30 peptide residues show chemical shifts in the range of random coil values, thus excluding the presence of ␣-helix as well as of ␤-sheet conformations in both solution conditions. Only Val 18 exhibited a down-field shift, as expected for residues preceding a proline (30). Furthermore, the absence of typical medium and long range NOE patterns excluded the presence of either ␣-helix or ␤-sheet in EX30 in water and TFE solutions.
To investigate the presence of other kind of folded conformations (␤-turns, ␥-turns) important support came from other NMR parameters such as low temperature coefficients and specific NOEs connectivity. The occurrence of low temperature coefficients (Ͻ4.0 ϫ 10 Ϫ3 ppm K Ϫ1 ) for the amide protons have been attributed to hydrogen bonding of the amide proton to the peptide backbone (31). In the case of ␤-turns the lowered temperature coefficient is due to the presence of a hydrogen bond between the NH of residue iϩ3 of the turn with the CϭO of residue i. A value for the temperature coefficient in the range of 4.0 -5.0 is usually attributed to a very weak H-bond or to an equilibrium between turn and other conformations without an H-bond (32). In H 2 O/D 2 O the EX30 peptide shows, for nonoverlapping residues, very high (Ͼ7.5 ppb/K) temperature coefficients, thus excluding the presence of H-bonds. For EX30 peptide in TFE solution, some relatively low amide proton temperature coefficients (4.0 Ͻ Ϫ␦⌬/␦TϽ 5.0) were measured for residues Leu 2 , Leu 8 , Gly 15 , Val 21 , and Gly 23 , suggesting the presence of some turn conformations (Fig. 3A). These were further confirmed by the presence of typical ␦ ␣N (i,iϩ2) NOE connectivities. The existence of this typical medium range NOE is a good indication of a significant population of turn conformations present in solution (33). In TFE, the occurrence of ␦ ␣N (i,iϩ2) NOE connectivities between Ala 6 /Leu 8 , Val 13 /Gly 15 , Pro 19 /Val 21 , and Val 21 /Gly 23 , together with strong ␦ NN (i,iϩ1) NOE connectivities point to the existence of different turn structures in rapid inter-conversion with open conformations (Fig. 3A). In water the EX30 peptide shows only intra-residue and sequential NOE connectivities, suggesting the presence of only extended conformations (data not shown). The assignment of extended conformations is also consistent with the weak intraresidue ␦ ␣N NOEs observed, compared with the strong NOE of sequential ␦ ␣N (i,iϩ1) protons (Fig. 3B, top) (34). The reduced intensity of ␦ NN sequential NOE cross-peaks is also typical of extended conformations (Fig. 3B, bottom). Distinguishing between the two extended conformations, PPII and ␤-strands, by NMR spectroscopy is not straightforward. PPII and ␤-strand both belong to the upper left corner of the Ramachandran map, with small differences in their dihedral angles (PPII, ϭ Ϫ78, ϭ 150; ␤-strand, ϭ Ϫ120, ϭ 113). The torsion angles and are essential parameters for defining the backbone conformation of a polypeptide chain. Among these, the angle can be evaluated by measuring the 3 J (HN-H␣) coupling constants, to which this angle is related. The 3 J (HN-H␣) coupling constants were measured from the one-dimensional spectra, and the extracted values were used for calculating dihedral angle , applying the Karplus equation (35) as parameterized by Vuister and Bax (36). While this NMR parameter is particularly useful in the case of highly stable and regular secondary structures such as ␣-helix and ␤-sheet, it is less informative in the case of flexible and not regular peptides (37). Values for 3 J (HN-H␣) between 6 and 7.5 Hz are usually attributed to random conformations or PPII conformations, while values above 8 Hz are usually considered strong indication of ␤-strand. Nevertheless where the 3 J (HN-H␣) coupling constant could be measured, values were generally found to be larger than the random coil values. This trend toward higher values was more pronounced when the temperature was increased, pointing to a thermal transition toward the ␤-strand conformation, as was also suggested by CD difference spectra.
FTIR Spectra-The deconvoluted FTIR spectrum of the amide I and II regions of lyophilized EX30 peptide is shown in Fig.  4. The amide I region contains two main components at 1671 cm Ϫ1 and at 1651 cm Ϫ1 . The first one is usually assigned to non-hydrogen-bonded groups or groups weakly bonded to the solvent, normally absorbing at 1666 to 1670 cm Ϫ1 (38). In our case, because of the absence of the solvent, the band is essentially due to the presence of PPII conformation (39,40), which is an extended structure lacking intramolecular hydrogen bonds (20,29). Furthermore, the component at 1651 cm Ϫ1 is assigned to unordered conformations and/or ␣-helical conformations (41). The amide I region also presents two small bands at 1633 cm Ϫ1 and at 1693 cm Ϫ1 indicative of cross-␤ and antiparallel ␤-sheet structures, respectively (42)(43)(44). We propose for the EX30 lyophilized peptide a dominance of PPII conformation together with unordered structures, with ␤-structures being of minor contribution. This interpretation is supported by the appearance of the band at 1549 cm Ϫ1 in the amide II (Fig. 4) region, which was attributed to PPII conformation in poly(GGA). This polymer has repeating triplets of the GGX type (45) similar to those found in the EX30 sequence. Furthermore, the presence of small amounts of ␤ conformation is confirmed by the band at 1524 cm Ϫ1 , which is normally attributed to the dominance of the bending of hydrogen bonded NH groups in antiparallel ␤-sheet structures (46). FTIR analysis of the Amide I region of EX30 fibers (Fig. 5) shows a prominent band at 1629 cm Ϫ1 due to antiparallel ␤-sheets of the cross-␤ type, as assigned above, together with a small band at 1697 cm Ϫ1 , which is typical of the antiparallel ␤-sheet conformation. Two minor bands at 1649 and 1673 cm Ϫ1 could originate from unordered and PPII conformations, respectively. The deconvoluted amide II region of EX30 fibers (Fig. 5) shows two components at 1516 cm Ϫ1 as well as at 1558 cm Ϫ1 that are considered representative of the presence of ␤-sheet conformation (45,46). The band at 1542 cm Ϫ1 could be tentatively assigned to the unordered component. Together, the FTIR data clearly show that the antiparallel ␤-sheet is the main structural component of EX30 fibers, whereas in the lyophilized EX30 peptide it is of minor contribution because of the dominance of PPII and unordered conformations. The amide A analysis of both peptides is shown in Figs. 6 and 7. It is evident the presence of one strong band at 3302-3303 cm Ϫ1 , which is also present in collagen and elastin (47), which both contain the PPII conformation. In addition, it is to be noted that the amide A of lyophilized EX30 shows the presence of significant absorptions originating from the OH stretching of water at 3491 and 3562 cm Ϫ1 , which are negligible in the case of EX30 fibers. A possible interpretation may lie in the fact that fiber formation is a strongly hydrophobic process giving rise to a sudden segregation of the ␤-structured polypeptide chains from the aqueous environment. In contrast, lyophilization is a slow process allowing the water inclusion into the polypeptide chains.
Time-dependent Fibril Formation and Congo Red Dye Binding-The EX30 peptide (1.0 mM) in Tris buffer (50 mM), NaCl (1.5 M), CaCl 2 (1 mM) (pH 7.0) solution formed a gel-like material after 12 h at room temperature (Fig. 8). The formation of viscous material is indicative of the formation of amyloid-like fibrils (48,49). The interaction of this material with Congo Red dye is shown in Fig. 9 and provides additional evidence for the ability of EX30 to self-assemble to form amyloid-like structures. Congo Red is widely used as an indicator of amyloid deposition. The binding interaction is characterized by both hyperchromic (absorbance increase) and bathochromic (redshift) effects in the absorbance spectrum of the dye (50).
Microscopy Studies-Peptides derived from EX30, solubilized in physiological solution, and warmed up to 50°C were analyzed by negative staining and rotary shadowing electron microscopy and by scanning force microscopy. After a few minutes incubation at 50°C, the EX30 peptide formed 20 -25-nm-wide globules and 10 -12-nm-thick filaments of variable length, which tended to form sheets on the substrate. For incubation times longer than 48 h, the majority of peptides were organized into rather rigid filaments of various length and thickness which had the appearance of twisted ropes (Fig. 10, a-d). Their thickness varied from about 20 to 80 nm, and the pitch of the helix varied from 25 to 110 nm depending on the filament thickness. By scanning force microscopy, these elements revealed a rather complex organization (Fig. 11, a-f). Short and rigid filaments were still observed (Fig. 11a), which tended to form latero-lateral aggregates (Fig. 11d). In Fig. 11e a filament segment consisting of three globules can be resolved into a complex set of elements whose organization suggests a precise molecular orientation and ordered interconnections. Fig. 11f clearly shows that each filament, about 20 nm thick, consists of discrete elements that form an ordered array and contact similar elements on the adjacent filament, thereby giving rise to a composite filament about 60 nm thick. DISCUSSION The results obtained in this study show that the sequence encoded by exon 30 of human tropoelastin is able to give rise to an aggregation process induced by microenvironmental changes such as temperature and time. The end product of the aggregation is the formation of fibrils with an amyloidlike structure.
At the molecular level, the structure adopted by the EX30 peptide is highly dependent on solvent conditions. In TFE, the peptide adopts a folded conformation characterized by several ␤-turns as suggested by CD. Accordingly, NMR data strongly indicate the presence of turns spanning the sequences 5 AAGL 8 , 12 GVGG 15 , 18 VPGV 21 and 20 GVGG 23 , the last two possibly belonging to the so-called sliding ␤-turns (51). Differently from TFE, in aqueous solution the peptide is mainly characterized by the presence, possibly in equilibrium, of PPII and ␤-sheet conformations. The ratio of these structures depends on temperature and/or concentration and time, with the PPII structure being preferred at low temperature (this study) and at low concentration (20) and the ␤-sheet conformation being favored by opposite conditions.
In addition, FTIR studies in the solid state allowed us to observe the conformational differences of EX30 both as a lyophilized powder and as fibers. The results obtained demonstrate that although the ␤-sheet and PPII generally appears in both samples, the antiparallel ␤-sheet conformation is chiefly found in fibers. In the lyophilized powder, PPII and unordered conformations predominate. The presence of antiparallel ␤-sheet chains, probably originating from cross-␤-structures, is similar to what was suggested for elastin and lamprin sequences that share the GGLGX motif tandem repeat found in the EX30 sequence (52,53). Different experimental and theoretical studies have indicated in a combination of electrostatic, polar, and hydrophobic interactions the driving force toward amyloid formation (54). The absence of any polar amino acid in the sequence suggests that, in the case of EX30, amyloid fiber formation is predominantly driven by hydrophobic interactions. By increasing the temperature and/or the concentration of the peptide the hydrophobic interactions are favored, thus determining the crucial transition to the ␤-sheet conformation, strictly related to the aggregated state.
The possibility that the formation of ␤-structures give rise to aggregation is consistent with the mechanism proposed for the formation of amyloid fibrils by proteins such as lysozyme and transthyretin. Specifically, the formation of fibrils is a consequence of an unfolding process, induced by changes in microenvironment, which leads to antiparallel alignment of cross ␤-structures that in turn give rise to the formation of fibrils and other aggregates that accumulate in the extracellular space (Ref. 55 and references therein). By contrast, this conformation is almost disfavored by the presence of proline residues that stabilize the PPII structure (29) and whose dihedral angle is frozen at a value (around Ϫ70°) not compatible with that of antiparallel ␤-structure (around Ϫ120°).
Quite interestingly, an increase of temperature and/or concentration also favors another type of self-aggregation, namely coacervation, although involving different sequences.
Coacervation is one of the peculiar properties of tropoelastin and has been proposed to promote alignment and cross-linking of tropoelastin molecules (56). It is a reversible self-aggregation process in which the protein separates from the solution as a second phase on increasing the temperature. In this case one can observe the formation of well ordered filamentous structures (57), while from a conformational point of view, coacervation promotes the formation of folded conformations, mainly ␤-turn conformations (58). The domains of human tropoelastin responsible for coacervation have been identified recently (28). Interestingly, these domains all belong to the larger prolinerich domains of tropoelastin (exon 18, exon 20, exon 24, and exon 26 coded domains). Finally, to coacervate the polypeptide sequences should be relatively long and possess a certain number of proline residues.
It has also been observed that polypeptide elastin-like sequences rich in proline, which undergo coacervation, are unable to form amyloid-like fibrils (9). Accordingly, the EX30 peptide gives rise to amyloid fibrils and does not coacervate but precipitates irreversibly (28). Although the role of proline residues in promoting the coacervation seems to be well established, there are polypeptide sequences encoded by the so-called KP domains of human tropoelastin that are unable to coacervate, even though they are rich in proline (data not shown). To this regard, we suggest that the peculiar propensity of isolated EX30 peptide to adopt a ␤-sheet conformation is the crucial element that favors  the adoption of amyloid-like organization rather than that expected for other elastin-like sequences.
Electron microscopy and atomic force microscopy of EX30 aggregates show the presence of fibrils that interact side by side and that could originate from an extensive self-interaction of elemental cross ␤-structures. Quite interestingly, the elastin-like biopolymer poly(VGGVG), which comprises the sequences LGGLG and VGGLG similar to those contained in EX30 peptide, has been shown to self-assemble in an amyloidlike pattern when deposited from aqueous suspensions (59). The fibrils did not solubilize after dilution, indicating the irreversibility of the process, also in this case.
Moreover, the sequence VGGLG is repeatedly found also in the prion protein, thus suggesting that this sequence could be involved in contributing to the self-assembly of amyloid fibers even in other proteins.
At this point, a question arises: the propensity of EX30 peptide toward amyloid fiber formation has a biological relevance for elastin and elastic fiber assembly?
Using an in vitro elastic fiber assembly system and parallel studies on transgenic mice where tropoelastin constructs were expressed, Kozel et al. (10) have evidenced the crucial role of C-terminal domains of tropoelastin for a correct elastic fiber formation. It has been suggested that EX30 domain is the major responsible for the interaction with microfibrils and that this interaction could be mediated by a mechanism of amyloidlike self-aggregation route.
Unquestionably, EX30 coded domain of tropoelastin is crucial for elastic fiber formation, as also indirectly demonstrated by the observation that all the different isoforms of tropoelastin contain it, but the way it happens has to be further investigated.
Our suggestion is that EX30 peptide, when inserted into the protein, does not favor amyloid-like aggregation but rather participates to the formation of filaments and bundles of filaments typical of the elastic fiber. Consistently, Miao et al. (8) demonstrated that in a polypeptide sequence constituted by different tropoelastin domain, the substitution of exon 24 domain with exon 30 domain did not alter the coacervation properties. On the contrary, when EX30 peptide is isolated from the protein it leads to the amyloid-like fibril organization reported in vitro in the present paper. Now, we would like to discuss, admittedly in a speculative manner, the possible relevance of these findings to some pathologies that affect elastic tissues (e.g. cardiovascular diseases and acute interstitial lung disease).
It is interesting to note that deposition of elastotic material in arteries occurs in aged, atherosclerotic humans, 2 and in lung alveoli in the case of acute interstitial lung disease. In the latter case, the elastotic material was shown to contain amyloid-like fibers (60). Furthermore, it is also known (61) that the degradation of human tropoelastin by elastase is strongly enhanced by the presence of unsaturated fatty acids, the same lipids found to accumulate on elastic fibers in atheromathous plaques. Accordingly, it is tempting to speculate that under certain pathological conditions, EX30derived soluble peptides released from elastin by proteolytic degradation could slowly aggregate because of mutated microenvironment to form amyloid-like structures that explain the amyloid "elastotic material" described in numerous reports. In fact, peptides PGVGGL and AGLGGLGVGGV, which belong to the EX30 sequence, have recently been identified in an elastase digest of insoluble human elastin (62). Therefore, EX30 peptide could play a role in the development and progression of pathological events that arise secondary to degradation of elastin.