Fibril formation in presence of cells or islets

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Monitoring the kinetics of fibril formation using Thioflavin T fluorescence


Thioflavin T (ThT) fluorescence is one of the “gold standards” techniques used to identify and study kinetics of amyloid fibrils. ThT chemical structure is presented in Fig. 22. The yellow-orange powder dye is a specific stain for amyloid fibrils in vivo and in vitro. It was first described in 1959 by Vassar and Culling as a fluorescence microscopy probe in histological samples for amyloid fibril deposits. Then in 1989 Naiki et al. found that the fibril concentration and emission intensity of ThT let to quantified amyloid fibrils in vitro. [97, 98]
This linearly correlation of ThT fluorescence with the amyloid fibrils has been probed in a range of 0.2 – 500 μM of Thioflavin T. [99]
Thioflavin T fluoresces slightly in the absence of amyloid fibrils, at the excitation of 350 and emission of 438 nm. Conversely there is a bright fluorescence in the existence of amyloid fibrils, at the excitation and emission maxima of 450 and 482 nm, respectively. [100] As the fluorescence increase occurs specifically once the Thioflavin T is binding to cross-β-sheet quaternary structure of amyloids, this probe allow to study the time-course of fibrillization. [101] presence of amyloid fibrils (adapted from M. Schlein 2017)
The specificity of ThT to identify amyloid fibrils is due to the property of ThT to bind only to this insoluble protein aggregation. The characteristic Cross-β structure of amyloid fibrils leaves a kind of cross-linked space in which the thioflavin molecule can bind (Fig. 23). The model of ThT binding to fibril-like β-sheets (Fig. 24) is suggested to be the intercalation of ThT along the surface side-chain channels running parallel to the long axis of the β-sheet. [102]
One application of ThT is kinetics of fibril formation. As shown in Figure 25, the kinetics of amyloid formation for hIAPP begins with a monomeric version of the polypeptide (lag phase), this peptide aggregation continues to form oligomers. The elongation phase represents the assembly of protofibrils into fibrils. The last phase, called saturation or plateau phase, ends with the mature fibrils. [66]

Experimental protocol for ThT T assay

Peptide synthesis and sample preparation

Mature hIAPP and the two flanking peptides were synthesized with a CEM Liberty Blue (CEM corporation, Matthews, USA) automated microwave peptide synthesizer using standard reaction cycles at the Institut de Biologie Intégrative (IFR83 – Sorbonne Université). The synthesis of mature hIAPP with an amidated C-terminus and a disulfide bridge was performed as described. [124] The synthesis of all peptides was performed using Fmoc chemistry and a PAL Novasyn TG resin. For mature hIAPP, two pseudoproline dipeptides were chosen for the synthesis Fmoc-Ala-Thr(ΨMe,MePro)-OH replaced residues Ala-8 and Thr-9, and Fmoc-Leu-Ser(ΨMe,MePro)-OH replaced residues Leu-27 and Ser-28. Double couplings were performed for the pseudoprolines and for the residues following the pseudoprolines and for every β-branched residue. The three peptides were cleaved from the resin and deprotected using standard TFA procedures with 1,2-ethanedithiol, water, and trisiopropylsilane as scavengers. The three peptides were purified by reverse phase high-performance liquid chromatography (HPLC) with a Luna C18(2) column (Phenomenex, USA). A two-buffer system was used. Buffer A consisted of 100% H2O and 0.1% TFA (vol/vol), and buffer B consisted of 100% acetonitrile and 0.07% TFA (vol/vol). Mature linear hIAPP was dissolved in aqueous DMSO (33%) and oxidized with air to the corresponding disulfide bond. Purity of peptides was higher than 95% as determined by analytical HPLC and identity of peptides was confirmed by MALDI-TOF mass spectrometry.
To evaluate aggregation kinetics of amyloid peptide is necessary to begin with the monomeric form of the peptide. Then, peptide stock solutions were freshly prepared prior to all experiments using the same batch. Peptide stock solutions were obtained dissolving the peptide at a concentration of 1 mM in hexafluoroisopropanol (HFIP) followed by 1 hour incubation. Then, HFIP was evaporated and the sample was dried by vacuum desiccation for at least 30 min. The resulting peptide film was dissolved at a concentration of 1 mM in DMSO for the fluorescence experiments (final DMSO concentration of 2.5% v/v) and then diluted in 20 mM Tris-HCl, 100 mM NaCl at pH 7.4. Both DMSO and NaCl interfere with the circular dichroism experiments, therefore in these experiments the peptide film was directly dissolved in a 20 mM sodium phosphate buffer, 100 mM NaF at pH 7.4. [124]

Kinetics of amyloid assembly

IAPP film peptide was completely rehydrated for 1 hour with Dimethyl sulfoxide (DMSO) to obtain the desired concentration of the peptide.
The fluorescence plate was filled with the buffer solution, ThT, DMSO and the peptide in the ratio necessary for the desired concentration. Fluorescence was monitored for a total time of approximately 40 hours. Amyloid formation was measured by thioflavin T fluorescence (λexcitation = 440 nm, λemission = 485 nm) with measurements every 10 minutes. The microplate was agitated for 10 seconds, fibrillisation kinetics were performed at 30 °C.
The buffer solution (20 mM tris, 100 mM NaCl, pH 7.4) and ThT solution (0.4 mM) were prepared before starting the experiment.
In order to initiate the fiber formation we added the hIAPP peptide at the end. Experiments of fluorescence were accomplish in triplicate in a 96-well Assay plate (COSTAR® 3792 Assay plate, no lid, round bottom, non-treated, non-sterile, black polystyrene using BMG FLUOstar OPTIMA Microplate Reader.

Identifying the change in the secondary structure of a protein with circular dichroism


Circular dichroism (CD) is a spectroscopy technique used for obtaining information of protein structure and conformation.
This technique is based in the interaction of polarized light with an asymmetric molecule (chiral). The light contains electromagnetic waves that travels in space and oscillate in time. The difference in the direction of light, make it unpolarized (in all directions) or plane polarized (one direction) as shown in Figure 26. For the circularly polarized light, the direction of rotation can be right (clockwise) or left (counterclockwise). [103 , 104]
The difference in the absorption of left-handed and right-handed circularly polarized light defines the circular dichroism property. [105]
The CD technique, use a series of prisms, mirrors, lens and filter to have plane polarized, then circularly polarized light that passes through the sample cell and gives a CD spectrum specific for the protein in study, as we can see in Fig. 6

Interpretation of the results of circular dichroism

The shape of the circular dichroism spectra curve offer information about the protein. The results of CD for proteins in the 180–250 nm UV region lead to identify the -helices, β-sheet or random coil conformations, characteristics of the secondary structure. [106] The « ‘w »-shaped spectra with troughs around 222 and 208 are representative of -helices structures, and a « ‘v »-shaped spectra with a trough at 217–220 nm is indicative of β-sheet structures, as shown in figure 28. This secondary structure conformation lets observe the changes of protein folding in solution. [107] Characteristic far-UV CD spectra for an all-a-helix, an all-β-sheet and a random coil protein. The spectrum for an all a-helix protein has two negative bands of similar magnitude at 222 and 208 nm, and a positive band at ~ 190 nm. The spectrum for an all β- sheet protein has in general a negative band between 210-220 nm and a positive band between 195 – 200 nm. The spectrum for a disorderly (random) protein has a negative band of great magnitude at around 200 nm. [188] CD is the differential absorption between left and right circularly polarized light of a chiral molecule. A chiral molecule has a non-superimposable mirror image configuration. The circular dichroism (CD) is the most commonly technique used to analyze the secondary structure of chiral biomacromolecules such as proteins.
These conformational changes can be measured, for example, during the protein aggregation process. This has an application in the study of pathologies of amyloid proteins such as Alzheimer’s or type 2 diabetes. [108]


Experimental protocol for CD

Circular dichroism spectroscopy

The peptide film was rehydrated at a final concentration of 25 µM just before starting the CD analysis with buffer solution of 20 mM phosphate, 100 mM NaF pH 7.4 Circular dichroism was performed on a Jasco 810 spectropolarimeter (Jasco Inc., Easton, MD) with a Peltier temperature-controlled at 25°C.
All spectra were measured from 190 to 260 nm in 0.1 cm path length quartz cuvettes (volume 200 µL) from Hellma GmbH, at 0.2 nm intervals and 10 nm.min-1 scan speed.
ϴ is the observed ellipticity (degrees) at wavelength 190-260 nm, N is the number of peptidic bound, c is the peptide concentration (dmol.L−1) and l is the path length cuvettes (cm).
The units of mean residue ellipticity are deg.
Then, we plotted the mean residue ellipticity against wavelength (nm), in order to identify the secondary structure of the peptide.

Observation of amyloid fibers with Transmission Electron Microscopy


Resolution is the distance at which two image points can be distinguished. The smallest distance between two points that a human eye can differentiate, is about 0.1–0.2 mm. To see smaller images than 0.1 mm we use a microscope. The most commonly know is the visible light microscope that magnifies images by optical lenses using visible light. In the electron microscopy techniques we use electrons as the illumination source. This increases the imaging resolution to 0.05 nm and magnifications of up to about 10,000,000x. When the electrons penetrate through the samples to form images, the instrument is a transmission electron microscope (TEM), to do this the sample must be thin, approximately less than 100 nm. This technique offers a high imaging resolution for structural and chemical composition information of the sample. [109, 110, 111]
The basic elements of a TEM (fig. 29) are the electron source (producing a stream of electrons) which is focused into an electron beam using condenser lenses. The electrons go through the sample and part of the electron beam is transmitted. The transmitted portion is focused by the objective lens creating the image. The image is passed down the column through the projector lenses, which are enlarged through each of the lens. TEM images are then projected onto a fluorescent screen. [110, 111]
The sample must be as thin as possible (≤ 100 nm), have all the conditions to conserve its original structure and work in a transmission electron microscope. The principally preparation method is TEM grid. These grids provide the support. A common type of grid is 3.05 mm in diameter, made normally of copper because of its cost and nonmagnetic property. Some grids are coated with support films, usually pure carbon because of its high mechanical strength, chemical stability with specimen and good electrical conductivity. In the figure Fig. 30 the different steps of the preparation of the sample are represented. [110]
The electrons bombarded on the sample generate various signals, as shown in Fig. 31 which are the emission of electrons with different energy spectra and other radiations. This provides information about the sample’s surface topography, crystallographic structure, chemical composition among others and various images of the sample can be obtained. [109, 110, 111]
Specifically for IAPP, the electron transmission microscopy technique offers valuable information, as confirmation of the formation of peptide fibrils. Normally, after the ThT fluorescence and CD results, it is the technique to be used to corroborate the fibril formation.

Experimental protocol for TEM technique

Transmission Electron Microscopy

TEM was performed at the “Institut de Biologie Paris Seine” (IBPS, Paris, France) at Sorbonne Université, Faculté de Sciences Campus Pierre and Marie Curie. Aliquots (20 µL) of the peptides in solution at 25 µM used for fluorescence assays were removed at the end of each kinetic experiments, blotted on a glow-discharged carbon coated 200 mesh copper grids for 2 minutes and then negatively stained with saturated uranyl acetate for 45 seconds. Grids were examined using a JEOL electron microscope operating at 80 kV.
Studying the interaction of the membrane with the amyloid peptide with model membrane assays, the calcein fluorescent probe

Large unilamellar vesicles by extrusion technique (LUV)

The most used method for unilamellar liposomes preparation is extrusion. To obtain Large unillamelar vesicles (LUV’s) by the by extrusion technique, lipids are in a chloroform solution and then dried to form a lipid film. The lipid film is hydrated with an aqueous buffer, heated to a temperature above the lipid phase transition temperature, and agitated.
To obtain the LUV, the solution is passed through a track etched polycarbonate membrane of a well-defined pore size (Fig. 32 Avanti’s Mini-Extruder for LUV Preparation). [112, 113]

Table of contents :

Chapter 1 Introduction to amyloid proteins and disease
1.1 Protein folding and misfolding
1.2 Protein folding
1.3 Amyloid Proteins
1.4 Amyloid fibril structure and amyloid formation
1.5 Factors of protein aggregation
1.6 Diabetes mellitus
1.7 The insulin secretory granule
1.8 IAPP expression
1.9 IAPP Post-translational modification
1.10 Physiological functions of IAPP
1.11 IAPP amyloidogenesis and structure
1.12 β-cell failure in type 2 diabetes
1.13 Apoptosis
1.14 hIAPP toxicity
1.15 Primlintide, an IAPP analogue
1.16 IAPP research
1.2 Objectives of the thesis
Chapter 2 Materials and methods
Biophysical methods
Monitoring the kinetics of fibril formation using Thioflavin T fluorescence
2.1.1 Principle
2.1.2 Advantages, drawbacks and requirements for ThT T assay
2.1.3. Experimental protocol for ThT T assay
Identifying the change in the structure of a protein with circular dichroism (CD)
2.2.1 Principle
2.2.3. Experimental protocol for CD
Observation of amyloid fibers with Transmission Electron Microscopy
2.3.1 Principle
2.3.2 Advantages, drawbacks and requirements for TEM technique
2.3.3 Experimental protocol for TEM technique
Studying the interaction of the membrane with the amyloid peptide with model membrane assays, the calcein fluorescent probe
2.4.1. Principle
2.4.2 Advantages, drawbacks and requirements for preparation of Large unilamellar vesicles by extrusion technique (LUV) and Calcein assay
2.4.3. Experimental protocol
Cell biological techniques
Confirming cytotoxicity with a cell viability test, The MTT Reduction Assay
2.5.1 Principle
2.5.2. Advantages, drawbacks and requirements for MTT assay
2.5.3 Experimental protocol for MTT assay
Chapter 3 The flanking peptides issues from the maturation of the human islet amyloid polypeptide (hIAPP) do not modify hIAPP-fibril formation nor hIAPP-induced cell death
3.1 Introduction
3.2 Materials and Methods
3.2.1 Materials
3.2.2 Peptide synthesis and preparation
3.2.3 Determination of peptide aggregation by thioflavin-T assay
3.2.4 Transmission Electron Microscopy (TEM)
3.2.5 Circular dichroism
3.2.6 Membranes preparation
3.2.7 Vesicle Dye Leakage Assay
3.2 8 Cell culture
3.2 9Human islet culture
3.2.10 Fibril formation in presence of cells or islets
3.2.11 MTT Cell Toxicity Assay
3.2.12 Statistics
3.3 Results and discussions
3.3.1 The flanking peptide are not amyloidogenic in solution
3.3.2 Do the flanking peptides influence mature hIAPP fibrillation in membrane models, in cells and in human islets?
3.4 Discussions
Chapter 4 β-pancreatic amyloid deposit, a protein conformational disease involved into type 2 diabetes: deleterious role of plasma membrane lipids
4.1 Kinetics of hIAPP fibrillation in presence of different cell lines
4.2 Determination of hIAPP toxicity in presence of different cell lines
4.3 Implication of IAPP receptor
4.4 Role of lipids membrane on hIAPP fibrillation
4.5 Effect of a diabetic environment on hIAPP fibrillation
4.6 hIAPP fibrillation ion human islets
V. Discussion of results


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