Resultados Cualitativos

We used a computer-based algorithm known as TANGO to predict the amyloidogenic propensity of substance P. All the predictions were conducted at pH 7, 25°C and ionic strength of 0.15M which aligns with conditions used for in vitro studies. Figure 3.1(A) shows the percentage of amyloidogenic propensity against the amino acid residues of substance P.

Figure 3.1: Substance P self-assembly. (A) TANGO analysis: amyloidogenic propensity and aggregation-prone

regions. (B) ThT binding assay of 1% w/w substance P in 150 mM NaCl.

A high percentage of amyloidogenic propensity was shown by the amino acid residues FFGLM in substance P sequence. This result indicates that these residues are more prone to aggregate at the conditions studied. In contrast, Singh et al reported that substance P has zero potential to form amyloid-like structures using the same analysis [27]. However, this study has used a lower peptide concentration compared to the present study. Since peptide self-assembly de highly depends on peptide concentration that might be the reason for this discrepancy.

Thioflavin T (ThT) binding fluorescence assays were used to study the self-assembling kinetics of SP in 150 mM NaCl following the protocol described in Chapter 2, section 2.2.1. Amyloid

66 specific dye ThT binds to the cross β-sheet structure of amyloids [21, 22]. ThT exhibits a significant increase in fluorescence intensity after binding to the cross β-sheet structure. The self-assembling process of 1% (w/w) SP in 150 mM NaCl was monitored at different time intervals and fitted to a single exponential curve (Figure 3.1B). The experiment was carried out in triplicates. Even though, at the beginning of the experiment (t0), low ThT binding was

observed, after 1 hour an increase in ThT fluorescence was observed, which reached the maximum at approximately 4 hours and then remained constant. The rate constant of aggregation was calculated from the best fit of the kinetic traces by single exponential curves (first-order kinetics), as previously reported [28] and detailed in section 2.2.1. Table 3.1 shows the comparison of lag phase and rate constants of aggregation (Kagg) calculated in 150 mM NaCl for substance P with that of LHRH (section 3.4.1) and somatostatin-14 (section 4.3).

Table 3.1: Kinetic parameters calculated for substance P, somatostatin and LHRH in 150 mM NaCl.

Peptide Lag phase Kagg (s-1)

1% w/w Substance P 0 2.33×10-4_±0.28×10-4

1% w/w Somatostatin 8 hours 0.09×10-4_±0.02×10-4

3% w/w LHRH 5 days 0.05×10-4_±0.01×10-4

According to table 3.1 data, among the three neuropeptides studied substance P has the highest rate constant of aggregation. Importantly, substance P does not show a lag phase prior to aggregation, whereas both somatostatin and LHRH show a lag phase prior to the aggregation. Apart from somatostatin and LHRH, functional amyloids such as β-endorphin and glucagon- like peptide-1 have also been shown to exhibit a lag phase prior to the aggregation [3, 6, 29]. The increase in ThT fluorescence indicates the presence of β-sheet rich structures in solution. Similar results were reported by Maji and co-workers previously for SP self-aggregation kinetics accessed by ThT assay, however in the presence of heparin [27]. The absence of a lag phase, the higher rate constant of aggregation and increase ThT fluorescence over the time indicate that substance P rapidly forms β-sheet based oligomers under the conditions studied. Notably, ThT results reveal that substance P can also self-assemble in the absence of an aggregation helper.

67 All SP acetate samples in 150 mM NaCl were observed to form gels from 5% w/w onwards. Optical microscopy under cross polarizers was performed to reveal whether these gels possess liquid crystalline structures following the protocol described in Chapter 2, section 2.2.3. All SP gels exhibit birefringence under crossed polarizers, showing that anisotropic structures are formed. Figure 3.2 shows different liquid crystalline textures observed for substance P in 150 mM NaCl.

Figure 3.2: Liquid crystalline optical textures formed by substance P (magnification ×10). (A) Threaded-

like textures. (B) cholesteric focal conic defects. Note: sample concentration - 10% w/w SP in 150 mM NaCl, sample age- two weeks old.

Two types of liquid crystalline optical textures were observed under cross polarizer for pure SP self-assemblies without any specificity to peptide concentration (Figure 3.2). Figure 3.2A shows threaded like textures which result from local and periodic variations in molecular orientation, and generally arise from nematic phases [30]. This type of textures have previously been observed for somatostatin-14 assemblies formed in both water and 150 mM NaCl [1]. Figure 3.2B exhibits cholesteric focal conic defects which were also previously reported for amyloid systems [31, 32]. These textures are compatible with columnar hexagonal liquid- crystal phases where molecules assemble into rod-like structures [33, 34]. These observations demonstrate the liquid crystalline nature of the SP self-assemblies. SP hence spontaneously self-assembles at room temperature into liquid crystalline phases with nematic properties, in 150 mM NaCl.

68 Using Fourier-transform infrared spectroscopy (FTIR), amide I vibrations (1600-1700 cm-1₎

can be used to identify the secondary structure of the peptide backbone. Amide I vibrations mainly arise from vibrational stretching modes of the backbone carbonyl groups. They can hence reveal information about the intermolecular and intramolecular H-bonds in which the backbone carbonyl groups are involved [35, 36]. The attenuated total reflectance (ATR) FTIR spectra were recorded at room temperature for SP self-assemblies in 150 mM NaCl, following the protocol described in section 2.2.2. Self-assembly of SP at three different concentrations (5%, 10%, and 20%) was followed by IR spectroscopy until 1 week. Figure 3.3 shows amide I region of all three concentration of SP studied at the end of one week.

Figure 3.3: ATR-FTIR spectra of mesophases of SP acetate in 150 mM NaCl as a function of peptide acetate concentration: from bottom to top, respectively 5%, 10% and 20%w/w. Note: Samples are one week old

The spectra showed unstructured amide I vibrations at low concentrations (5%, 10%) which evolved towards well-structured amide I vibrations at higher peptide concentrations (20%w/w). The structured amide I of 20%w/w SP could be assigned into four well-defined vibrations at 1622 cm-1 _{and 1673 cm}-1 _{for antiparallel β sheet hydrogen-bonds, 1633 cm}-1 _{for parallel β sheet}

hydrogen-bonds and 1652 cm-1 _{for random coil conformations as previously reported [1].}

Similar vibrations were observed for 10%w/w SP assemblies as well. However, 5%w/w SP assemblies only exhibit vibrations at 1622 cm-1 _{and 1652 cm}-1 _{that corresponds to major peak}

for antiparallel β-sheet and random coil, respectively. In other words, 5% w/w SP assemblies comprise of the random coil to a greater extent. This observation strongly suggests that the structuration of amide I vibrations is directly correlated to the peptide concentration, supporting

69 that more secondary structures are formed at higher concentrations. In accordance with the literature, antiparallel β sheets (1622 and 1672 cm-1_{) network is due to the presence of}

nanofibrils in solution [1]. On the other hand, vibration at wavenumbers of around 1630 cm-1

is generally assigned to a parallel β sheet network. According to the previously published reports, 1630 cm-1 _{is not related to the nanofibrils formation but correlated to the lateral}

association of nanofibrils into nanofibers [37]. A similar vibration was previously reported for Somatostatin-14 in the water at 1627 cm-1 _{which has been assigned for parallel β sheet network}

[1]. To determine the number of backbone carbonyls involved in each of these vibrations, we decomposed the FTIR spectra obtained for 20%w/w into individual components (Table 3.2).

Table 3.2: Percentages of amyloid vibrations within amide I region after deconvolution of SP 20%w/w in NaCl FTIR spectra using the Opus software (Bruker).

Given the presence of 11 backbone carbonyls in the SP sequence, 7 backbone carbonyl groups are involved in the antiparallel β sheet networks, 2 carbonyls in parallel β sheet networks and 2 carbonyls in random conformation, on average. This type of calculation is only valid if all the peptide molecules are involved in the same structure. In other words, all molecules should contribute to fibril architecture and should not be any oligomers present in the solution. To minimize the effect of oligomers, present in the solution, we calculated these values for highest peptide concentration (20% w/w) studied.