# Real-Time  Monitoring of Apoptosis by Caspase-3-Like Protease Induced FRET Reduction Triggered by Amyloid Aggregation

Authors: Johan F. Paulsson; Sebastian W. Schultz; Martin  Köhler; Ingo Leibiger; Per-Olof Berggren; Gunilla T. Westermark
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Amyloid formation is cytotoxic and can activate the caspase cascade. Here, we monitor caspase-3-like activity as reduction of fluorescence resonance energy transfer (FRET)  using the contstruct  pFRET2-DEVD containing enhanced cyan fluorescent protin (EYFP) linked by the caspase-3 specific cleavage site residues DEVD. Beta-TC-6 cells were transfected, and  the fluoorescence was measured at 440 nm excitation and 535 nm (EYFP) and 480 nm (ECFP) emission wavelength. Cells were incubated with recombinant pro lset Amyloid Polypeptide (rec prolAPP) or the processing metabolites of prolAPP;   the N-terminal flanking peptide withIAPP (recN+IAPP); IAPP with the C-terminal flanking peptied (recIAPP+C) and lslet Amyloid  Polypeptide (recIAPP) . Peptides were added in solubilized from (50 μM) or as performed amyloid-like fibrils, or as a combination of these. FRET was measured and incubation with a mixture of solubilized peptide and performed fibrils resulted in loss of  FRET and apoptosis was determined to occure in cells incubated with recproIAPP (49%), recN+IAPP (46%), recIAPP (72%) and recIAPP+C  (59%). These results show that proIAPP and the processing intermediates reside the same cell toxic capacity as IAPP, and they can all have a central role in the reduction of beta-cell number in type 2 diabetes.


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## Body

## 1. Introduction

If proteins misfold or lose
their
native fold,
they are usually assisted to refold by molecular chaperons or they can be
removed from the system and be degraded [1].  However, under certain circumstances this
system seems to fail and proteins escape degradation. Instead, they can
aggregate into fibrils with a high degree of beta-pleated secondary structure,
and if these fibrils are stained by Congo red and exert green birefringence
when viewed in polarised light they are referred to as amyloid [2–5]. Amyloid
deposition is associated with loss of organ function and cell death. Hitherto,
at least 25 different proteins have been characterised from systemic or localised
amyloid deposits in humans [4].The amyloid formation process can be divided into two phases. The first
is a nucleation forming process where monomers of the amyloid peptide form prefibrillar
oligomeric species and this is referred to as the lag phase [6, 7]. The second phase is the extension phase where rapid elongation of
amyloid fibrils occurs which will reach a plateau when most of the molecules are
converted into the fibrillar form. A dramatic reduction of the lag phase will
occur if mature amyloid fibrils of the same origin are added and this is called
the seeding effect. There is growing evidence that the cytotoxic species of the
amyloid forming process are early oligomers consisting of 15–40 monomers forming
a ring shaped structure [8–10]. These prefibrillar assemblies are able to incorporate and form pores in
cellular membranes causing cell leakage and influx of cations which triggers the
apoptosis cascade. According to this, the amyloid fibril itself is a nontoxic end
product of a cytotoxic aggregation process.Amyloid deposits in the islets of Langerhans are a pathological
characteristic of type 2 diabetes, and the amyloid aggregates consist of islet
amyloid polypeptide (IAPP)  [11, 12]. IAPP is a product of the endocrine beta-cell and as amyloid deposition
occurs a large reduction of beta-cell mass has been observed [13–15]. IAPP has also been shown to form spherical structures with the capacity
to incorporate into lipid bilayers [16–18]. In vitro studies of
different cell lines show that amyloid formation of IAPP is cytotoxic and
triggers the apoptotic pathway by activation of the caspase cascade [19–21].IAPP is expressed as a precursor molecule (proIAPP) which is posttranslationally
modified by the processing enzymes prohormone convertase (PC) 2 and 1/3 [22–25]. In the secretory granule, proIAPP is cleaved between dibasic residues
at the N- and C-termini by PC2 and PC1/3, respectively. We and others have
previously shown in cell lines that if aberrant processing of human proIAPP occurs,
intracellular amyloid-like aggregates arise with cell death as a result [26, 27]. Also intracellular amyloid-like
aggregates consisting of proIAPP have been described in both transgenic mice expressing
human IAPP and in human beta-cells [28]. Cells with intracellular amyloid-like
material are also positive for M30 cyto-death antibody which binds to an early neoepitope
that becomes accessible during caspase activation.Here, we describe a novel system for monitoring beta-cell apoptosis. The
system uses two fluorophores linked together with a caspase 3-like cleavage
site, and when cleavage occurs a reduction of fluorescence resonance energy
transfer (FRET) can be measured as an
indicator of apoptosis. Since measurements of the same cells can be done
repeatedly, real-time
monitoring of beta-cell
apoptosis can be performed. Also, we used the established assay to compare the apoptotic
properties of recombinant proIAPP (recproIAPP)
and proIAPP processing intermediates, N-terminal flanking peptide with IAPP (recN+IAPP), IAPP with the C-terminal
flanking peptide (recIAPP+C), and recombinant IAPP (recIAPP).


## 2. Experimental Procedures

### 2.1. Cell Transfection

Beta-TC-6 (B-TC-6)
cells obtained from American Type Culture Collection (Manassas, VA, USA) were
cultured to 80% confluency in 10 cm diameter Petri dishes (Falcon: Labora, Stockholm,
Sweden) in RPMI-1640 medium with 11 mM D-glucose containing 10% fetal bovine
serum (FBS) (Sigma, Stockholm, Sweden), 100 IU/ml penicillin, 100μg/ml streptomycin, 50 μM β-mercaptoethanol. One
hour prior to transfection, medium was changed to RPMI-1640 medium without
serum. A total volume of 175 μL with
20 μg pFRET2-DEVD, 20 μg pcDNA3, 10 mM polyethyleneimine,
and 5% sucrose was added to 9 mL of RPMI-1640 medium without serum. After 6
hours of incubation, FBS was added to a final concentration of 10%. After 24
hours, 0.4 mg/mL G-418 antibiotics was added to the medium for selection of stabile B-TC-6 clones.


### 2.2. Assay Buffer

The
following solutions were investigated for autofluorescence: (1) Krebs-Ringer with hepes and glucose
(KRHG) (120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 0.5 mM KH2PO4, pH 7.4
with 2 mM D-glucose, 20 mM Hepes, and 200 nM adenosine). (2) Hank’s balanced salt solution
(HBSS) (5.4 mM KCl, 0.3 mM NaHPO4, 0.4 mM KH2PO4, 4.2 mM
NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.6 mM MgSO4, 137 mM NaCl, 5.6 mM D-glucose, pH 7.4). (3) RPMI-1640 medium without FBS
(Sigma). (4) RPMI-1640 medium with
10% FBS. (5) Dulbecco´s medium
without phenol red (Invitrogen, Carlsbad,
Calif, USA). (6) Schneider´s Drosophila medium without
phenol red (Invitrogen). (7) H20.


### 2.3. Real-Time Monitoring of Apoptosis

pFRET2-DEVD vector
is driven by the insulin promoter and can only be expressed in insulin
producing cells. When expressed, a product consisting of enhanced cyan
fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP)
linked by the amino acid residues DEVD will be dispersed throughout cytoplasm.
The sequence DEVD is a specific substrate for caspase-3-like proteases and when
ECFP and EYFP are connected by the DEVD residues FRET will occur. If the
transfected B-TC-6 cells undergo apoptosis, the DEVD is cleaved and the two
fluorophores will be separated and a loss of FRET can be monitored as a
decrease of the 535 nm/480 nm fluorescence ratio. The pFRET2-DEVD vector has previously
been characterised by Köhler et al.
[29]. B-TC-6 cells with stable expression pFRET2-DEVD were cultured for 48
hours in black 96 well plates (Labsystems) to 80% confluency and washed once in
KRHG-buffer prior to experimental procedures. Sample volume was set to 100 μL and both synthetic and recombinant peptides
were kept as stock solution of 5 mM in dimethyl sulfoxide (DMSO) and diluted to a
final concentration of 50 μM with a final DMSO concentration of 1%. Synthetic
human and rat IAPP were analysed in solubilised form at final concentrations of
25 and 50 μM. Negative control was 1% DMSO in KRHG-buffer and
when seeded assays were performed a final concentration of 30 nM synthetic IAPP fibrils
was included in the negative control. Positive control for apoptosis contained
2 μM staurosporine and 1% DMSO in KRHG-buffer. Negative and positive controls
were included in each individual assay. Mature recombinant amyloid-like fibrils
were washed in H2O and
centrifuged at 16.000 g for 15 minutes and resuspended in KRHG-buffer, sonicated
and diluted to an estimated concentration of 50 μM.Loss of FRET was measured in a Wallac 1420 multilabel
counter (Perkin Elmer, Turku, Finland) with WorkOut software version 1.5
(Perkin Elmer). Excitation was set to 440 nm, and emitted fluorescence was
measured at 535 nm (EYFP) and 480 nm (ECFP). Data are presented as mean ratio ±SEM of 535 nm/480 nm ratio. Each point represents at
least five individual measurements.


### 2.4. Confocal Microscopy

B-TC-6 cells with stabile expression of
pFRET2-DEVD and untransfected B-TC-6 cells were cultured on 19 mm cover slips
and rinsed in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and
1.4 mM, KH2PO4) before fixation in 2% paraformaldehyde in
PBS for 30 minutes. For visualisation, cells were incubated with the nuclear
stain TO-PRO-3 (Molecular probes, Eugene,
Ore, USA)
diluted 1 : 1000 in PBS for 15 minutes and then mounted with 50/50 glycerol/PBS. Cells
were studied in a Nikon eclipse E600 microscope with a Nikon C1 confocal unit
with argon 488 nm and HeNe 633 nm lasers (Nikon Kawasaki, Japan).
Digital pictures were taken with an EZ-C1 digital camera and software version
1.0 for Nikon confocal microscopy.


### 2.5. Production of Recombinant Peptides

Human preproIAPP cloned into the pBluescript II
vector was used as template for generation of PCR amplified DNA fragments
corresponding to human proIAPP, N-terminal flanking peptide+IAPP, IAPP and
IAPP+C-terminal flanking peptide. Forward and reverse primers used for this
purpose are described in Table1. The amplified IAPP fragments were blunt end ligated
in expression vector pGEX 2TK (GE healthcare, Uppsala, Sweden).
Constructs were confirmed by sequencing to be in correct reading frame and
without mutations. The pGEX 2TK vector has glutathione S-transferase (GST) in
front of the multiple cloning site and the peptide will be expressed as a GST
fusion protein. Y1090 bacteria were transformed and cultured in Luria broth at
+37∘C until OD A600 reached 0.8, and protein synthesis was induced with 3 mM isopropyl β-D-1
thiogalactopyranoside (IPTG) (Fermentas, St Lenon Rot, Germany) for 3 hours at
+25∘C. Bacteria were
spun down and resuspended in TEDG buffer (50 mM TRIS-HCl pH 7.4, 1.5 mM EDTA, 10%
glycerol, 400 mM NaCl) and sonicated three times for 20 seconds. The bacteria lysate was
centrifuged at 100.000 g in a SW41 Ti rotor for 30 minutes at +4∘C, and the supernatant was transferred to Sepharose-4B beads
(GE
healthcare) and incubated 2 hours end over end at +4∘C. Sepharose beads were spun down at 3000 g for one minute, and the
lysate was decanted and the beads were washed three times in NET-N buffer (50 mM
TRIS-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.5% NONIDET-NP 40 (USB, Cleveland, Ohio,
USA) followed by three times wash in PBS. The GST-tag was cleaved off with thrombin
protease (GE healthcare) 20 U/mg expected peptide in PBS end over end overnight.
Removal of the 27 kDa GST-tag resulted in rapid amyloid formation and aggregates
were collected. The amyloid-like fibrils were washed in ddH2O and
solved in 50%/50% 1,1,1,3,3,3-hexafluoro-2-propanol
(HFIP)/trifluoroacetic acid (TFA) for 24 hours. Solubilised peptides were
centrifuged at 16.000 g for 15 minutes, and the supernatant was recovered. For
assays with IAPP recombinant peptides in complete monomeric form, the
supernatant was filtered trough a Millex-FG syringe filter 0.2 μm for
hydrophobic solvents. Samples were dried in vacuum and redisolved in 100% DMSO.
Peptide concentrations for recproIAPP
and recN+IAPP were determined using A280 extinction coefficient
1615 M−1 cm−1and for recIAPP and recIAPP+C 3105 M−1 cm−1 in a nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del,
USA). Theoretical molecular masses were as follow: recproIAPP 8358 Da, recN+IAPP 6224 Da, recIAPP
4918 Da, and recIAPP+C 7053 Da. Synthetic
human IAPP used in the study was synthesized by KEX Laboratories, Yale University
(New Haven, Conn, USA), and synthetic rat IAPP was purchased from Bachem
(Heidelberg, Germany).Table 1
Forward
and reverse primers for amplification of IAPP fragments.
proIAPP forward5′-GAT GAC ACC CAT TGA AAG
TCA TCA GG-3′proIAPP reverse5′-CTA CTA AAG GGG CAA GTA
ATT CAG TGG-3′N-terminal+IAPP forward5′-GAT GAC ACC CAT TGA AAG
TCA TCA GG-3′N-terminal+IAPP reverse5′-CTA CTA GCC ATA TGT ATT GGA TCC CAC G-3′IAPP forward5′-GAT GAA ATG CAA CAC TGC
CAC ATG-3′IAPP reverse5′-CTA CTA GCC ATA TGT ATT GGA TCC CAC G-3′IAPP+C-terminal forward5′-GAT GAA ATG CAA CAC TGC
CAC ATG-3′IAPP+C-terminal reverse5′-CTA CTA AAG GGG CAA GTA
ATT CAG TGG-3′


### 2.6. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Tricine-SDS-PAGE was performed as described by Schägger
and von Jagow [30]. Samples were dissolved in sample buffer containing 0.1 M Tris-HCl  at pH 6.8, 30%
(wt/vol) glycerol, 8%
(wt/vol) SDS, 0.2 M dithiothreitol (DTT), and 0.02% Coomassie blue G-250 and
boiled for 5 minutes prior to loading onto the gel. The separation condition
was 30 mV for 20 hours.


### 2.7. Silver Staining and Trypsin Digestion

The tricine-SDS gel was sensitised in 1.4 mM Na2S2O4 and incubated in 0.2% AgNO3 solution containing 7.5 μL
formaldehyde (37%)/100 mL water for 25 minutes and developed in 6% Na2CO3, 25 μM Na2S2O3, and 50 μL
formaldehyde (37%)/100 mL water.Silver stained bands corresponding to the expected
masses of the recombinant IAPP peptides were excised from the gel and dried.
Each sample was incubated with 60μL reducing
agent consisting of 10 mM dithiothreitol (DTT) in 25 mM NH4HC3 for one hour
at +56∘C, after which the peptides were alkylated in 70 μL 55 mM iodoacetamide
(IAA) at room temperature in dark for 45 minutes. Rehydrated gel pieces were
digested in 100 μL trypsin
solution containing 1.5 μg/mL trypsin
(Promega, Madison, Wis, USA)
and 25 mM NH4HCO3 and incubated 24 hours at +37∘C.


### 2.8. Electro Spray Ionisation Tandem Mass Spectrometry

Full scan
mass spectra for peptide mass fingerprinting and TOF product scans for
sequencing of amino acids were acquired on a hybrid mass spectrometer (API
QSTAR Pulsar, Applied Biosystems, Foster city, Calif) equipped with a nanoelectrospray
ion source (MDS Protana, Odense, Denmark).


### 2.9. Amyloid Staining

Samples were dried onto glass slides and incubated
for 20 minutes in A solution (NaCl saturated 80% ethanol with 0.01% NaOH) and were
directly transferred to B solution (solution A saturated with Congo red).
Samples were rinsed in 100% ethanol and xylen and mounted. Amyloid specific
green birefringence was studied in an Olympus X51 microscope (Olympus, Tokyo, Japan)
with two polarising filters connected to an Olympus DP 50 digital camera run by
Studio Lite v 1.0.1 software.


### 2.10. Electron Microscopy

Droplets of sample were placed on formvar coated
copper grids and were negative contrasted with 2% uranyl acetate in 50% ethanol.
Samples were studied in a Jeol 1230
electron microscope at 100 kV (Jeol, Akishima, Tokyo, Japan).
Digital electron micrographs were taken with a Gatan multiscan camera model 791
with corresponding software v3.6.4 (Gatan Inc, Pleasanton, Calif, USA).


### 2.11. Thioflavin Tassay

Kinetic
studies of amyloid formation were performed in Sigmacote (Sigma-Aldrich, St.
Louis, Mo, USA) treated black 96 well plates (Labsystems) in a sample volume of
100μL. Synthetic IAPP was diluted from a DMSO stock
solution (5 mM) to a final concentration of 50 μM in thioflavin T
(ThT) in assay buffer (50 mM glycine, 25 mM sodium phosphate buffer, pH 7.0, and 10 μM ThT). Mature IAPP fibrils
(30 nM) were included
to decrease the amyloid lag phase. Samples consisting of ThT assay buffer with
1% DMSO and ThT assay buffer with 30 nM IAPP fibrils and 1% DMSO were included
in the assay. Fluorescence was measured every thirty minutes at an excitation
wavelength of 442 nm and an emission wavelength of 486 nm. Each sample was
measured in 12 individual wells, and data presented are mean values ±SEM.


### 2.12. Statistical Analysis

Statistics
were performed in GraphPad InStat version 3.06 (GraphPad Software Inc., San Diego, Calif,
USA). Unpairedt-test
was used when two groups were compared and one-way analysis of variance (ANOVA)
with Dunnett multiple comparisons test when several groups were analysed. A
P-value
less than 0.05 was considered significant.


## 2.1. Cell Transfection

Beta-TC-6 (B-TC-6)
cells obtained from American Type Culture Collection (Manassas, VA, USA) were
cultured to 80% confluency in 10 cm diameter Petri dishes (Falcon: Labora, Stockholm,
Sweden) in RPMI-1640 medium with 11 mM D-glucose containing 10% fetal bovine
serum (FBS) (Sigma, Stockholm, Sweden), 100 IU/ml penicillin, 100μg/ml streptomycin, 50 μM β-mercaptoethanol. One
hour prior to transfection, medium was changed to RPMI-1640 medium without
serum. A total volume of 175 μL with
20 μg pFRET2-DEVD, 20 μg pcDNA3, 10 mM polyethyleneimine,
and 5% sucrose was added to 9 mL of RPMI-1640 medium without serum. After 6
hours of incubation, FBS was added to a final concentration of 10%. After 24
hours, 0.4 mg/mL G-418 antibiotics was added to the medium for selection of stabile B-TC-6 clones.


## 2.2. Assay Buffer

The
following solutions were investigated for autofluorescence: (1) Krebs-Ringer with hepes and glucose
(KRHG) (120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 0.5 mM KH2PO4, pH 7.4
with 2 mM D-glucose, 20 mM Hepes, and 200 nM adenosine). (2) Hank’s balanced salt solution
(HBSS) (5.4 mM KCl, 0.3 mM NaHPO4, 0.4 mM KH2PO4, 4.2 mM
NaHCO3, 1.3 mM CaCl2, 0.5 mM MgCl2, 0.6 mM MgSO4, 137 mM NaCl, 5.6 mM D-glucose, pH 7.4). (3) RPMI-1640 medium without FBS
(Sigma). (4) RPMI-1640 medium with
10% FBS. (5) Dulbecco´s medium
without phenol red (Invitrogen, Carlsbad,
Calif, USA). (6) Schneider´s Drosophila medium without
phenol red (Invitrogen). (7) H20.


## 2.3. Real-Time Monitoring of Apoptosis

pFRET2-DEVD vector
is driven by the insulin promoter and can only be expressed in insulin
producing cells. When expressed, a product consisting of enhanced cyan
fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP)
linked by the amino acid residues DEVD will be dispersed throughout cytoplasm.
The sequence DEVD is a specific substrate for caspase-3-like proteases and when
ECFP and EYFP are connected by the DEVD residues FRET will occur. If the
transfected B-TC-6 cells undergo apoptosis, the DEVD is cleaved and the two
fluorophores will be separated and a loss of FRET can be monitored as a
decrease of the 535 nm/480 nm fluorescence ratio. The pFRET2-DEVD vector has previously
been characterised by Köhler et al.
[29]. B-TC-6 cells with stable expression pFRET2-DEVD were cultured for 48
hours in black 96 well plates (Labsystems) to 80% confluency and washed once in
KRHG-buffer prior to experimental procedures. Sample volume was set to 100 μL and both synthetic and recombinant peptides
were kept as stock solution of 5 mM in dimethyl sulfoxide (DMSO) and diluted to a
final concentration of 50 μM with a final DMSO concentration of 1%. Synthetic
human and rat IAPP were analysed in solubilised form at final concentrations of
25 and 50 μM. Negative control was 1% DMSO in KRHG-buffer and
when seeded assays were performed a final concentration of 30 nM synthetic IAPP fibrils
was included in the negative control. Positive control for apoptosis contained
2 μM staurosporine and 1% DMSO in KRHG-buffer. Negative and positive controls
were included in each individual assay. Mature recombinant amyloid-like fibrils
were washed in H2O and
centrifuged at 16.000 g for 15 minutes and resuspended in KRHG-buffer, sonicated
and diluted to an estimated concentration of 50 μM.Loss of FRET was measured in a Wallac 1420 multilabel
counter (Perkin Elmer, Turku, Finland) with WorkOut software version 1.5
(Perkin Elmer). Excitation was set to 440 nm, and emitted fluorescence was
measured at 535 nm (EYFP) and 480 nm (ECFP). Data are presented as mean ratio ±SEM of 535 nm/480 nm ratio. Each point represents at
least five individual measurements.


## 2.4. Confocal Microscopy

B-TC-6 cells with stabile expression of
pFRET2-DEVD and untransfected B-TC-6 cells were cultured on 19 mm cover slips
and rinsed in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, and
1.4 mM, KH2PO4) before fixation in 2% paraformaldehyde in
PBS for 30 minutes. For visualisation, cells were incubated with the nuclear
stain TO-PRO-3 (Molecular probes, Eugene,
Ore, USA)
diluted 1 : 1000 in PBS for 15 minutes and then mounted with 50/50 glycerol/PBS. Cells
were studied in a Nikon eclipse E600 microscope with a Nikon C1 confocal unit
with argon 488 nm and HeNe 633 nm lasers (Nikon Kawasaki, Japan).
Digital pictures were taken with an EZ-C1 digital camera and software version
1.0 for Nikon confocal microscopy.


## 2.5. Production of Recombinant Peptides

Human preproIAPP cloned into the pBluescript II
vector was used as template for generation of PCR amplified DNA fragments
corresponding to human proIAPP, N-terminal flanking peptide+IAPP, IAPP and
IAPP+C-terminal flanking peptide. Forward and reverse primers used for this
purpose are described in Table1. The amplified IAPP fragments were blunt end ligated
in expression vector pGEX 2TK (GE healthcare, Uppsala, Sweden).
Constructs were confirmed by sequencing to be in correct reading frame and
without mutations. The pGEX 2TK vector has glutathione S-transferase (GST) in
front of the multiple cloning site and the peptide will be expressed as a GST
fusion protein. Y1090 bacteria were transformed and cultured in Luria broth at
+37∘C until OD A600 reached 0.8, and protein synthesis was induced with 3 mM isopropyl β-D-1
thiogalactopyranoside (IPTG) (Fermentas, St Lenon Rot, Germany) for 3 hours at
+25∘C. Bacteria were
spun down and resuspended in TEDG buffer (50 mM TRIS-HCl pH 7.4, 1.5 mM EDTA, 10%
glycerol, 400 mM NaCl) and sonicated three times for 20 seconds. The bacteria lysate was
centrifuged at 100.000 g in a SW41 Ti rotor for 30 minutes at +4∘C, and the supernatant was transferred to Sepharose-4B beads
(GE
healthcare) and incubated 2 hours end over end at +4∘C. Sepharose beads were spun down at 3000 g for one minute, and the
lysate was decanted and the beads were washed three times in NET-N buffer (50 mM
TRIS-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA and 0.5% NONIDET-NP 40 (USB, Cleveland, Ohio,
USA) followed by three times wash in PBS. The GST-tag was cleaved off with thrombin
protease (GE healthcare) 20 U/mg expected peptide in PBS end over end overnight.
Removal of the 27 kDa GST-tag resulted in rapid amyloid formation and aggregates
were collected. The amyloid-like fibrils were washed in ddH2O and
solved in 50%/50% 1,1,1,3,3,3-hexafluoro-2-propanol
(HFIP)/trifluoroacetic acid (TFA) for 24 hours. Solubilised peptides were
centrifuged at 16.000 g for 15 minutes, and the supernatant was recovered. For
assays with IAPP recombinant peptides in complete monomeric form, the
supernatant was filtered trough a Millex-FG syringe filter 0.2 μm for
hydrophobic solvents. Samples were dried in vacuum and redisolved in 100% DMSO.
Peptide concentrations for recproIAPP
and recN+IAPP were determined using A280 extinction coefficient
1615 M−1 cm−1and for recIAPP and recIAPP+C 3105 M−1 cm−1 in a nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del,
USA). Theoretical molecular masses were as follow: recproIAPP 8358 Da, recN+IAPP 6224 Da, recIAPP
4918 Da, and recIAPP+C 7053 Da. Synthetic
human IAPP used in the study was synthesized by KEX Laboratories, Yale University
(New Haven, Conn, USA), and synthetic rat IAPP was purchased from Bachem
(Heidelberg, Germany).Table 1
Forward
and reverse primers for amplification of IAPP fragments.
proIAPP forward5′-GAT GAC ACC CAT TGA AAG
TCA TCA GG-3′proIAPP reverse5′-CTA CTA AAG GGG CAA GTA
ATT CAG TGG-3′N-terminal+IAPP forward5′-GAT GAC ACC CAT TGA AAG
TCA TCA GG-3′N-terminal+IAPP reverse5′-CTA CTA GCC ATA TGT ATT GGA TCC CAC G-3′IAPP forward5′-GAT GAA ATG CAA CAC TGC
CAC ATG-3′IAPP reverse5′-CTA CTA GCC ATA TGT ATT GGA TCC CAC G-3′IAPP+C-terminal forward5′-GAT GAA ATG CAA CAC TGC
CAC ATG-3′IAPP+C-terminal reverse5′-CTA CTA AAG GGG CAA GTA
ATT CAG TGG-3′


## 2.6. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Tricine-SDS-PAGE was performed as described by Schägger
and von Jagow [30]. Samples were dissolved in sample buffer containing 0.1 M Tris-HCl  at pH 6.8, 30%
(wt/vol) glycerol, 8%
(wt/vol) SDS, 0.2 M dithiothreitol (DTT), and 0.02% Coomassie blue G-250 and
boiled for 5 minutes prior to loading onto the gel. The separation condition
was 30 mV for 20 hours.


## 2.7. Silver Staining and Trypsin Digestion

The tricine-SDS gel was sensitised in 1.4 mM Na2S2O4 and incubated in 0.2% AgNO3 solution containing 7.5 μL
formaldehyde (37%)/100 mL water for 25 minutes and developed in 6% Na2CO3, 25 μM Na2S2O3, and 50 μL
formaldehyde (37%)/100 mL water.Silver stained bands corresponding to the expected
masses of the recombinant IAPP peptides were excised from the gel and dried.
Each sample was incubated with 60μL reducing
agent consisting of 10 mM dithiothreitol (DTT) in 25 mM NH4HC3 for one hour
at +56∘C, after which the peptides were alkylated in 70 μL 55 mM iodoacetamide
(IAA) at room temperature in dark for 45 minutes. Rehydrated gel pieces were
digested in 100 μL trypsin
solution containing 1.5 μg/mL trypsin
(Promega, Madison, Wis, USA)
and 25 mM NH4HCO3 and incubated 24 hours at +37∘C.


## 2.8. Electro Spray Ionisation Tandem Mass Spectrometry

Full scan
mass spectra for peptide mass fingerprinting and TOF product scans for
sequencing of amino acids were acquired on a hybrid mass spectrometer (API
QSTAR Pulsar, Applied Biosystems, Foster city, Calif) equipped with a nanoelectrospray
ion source (MDS Protana, Odense, Denmark).


## 2.9. Amyloid Staining

Samples were dried onto glass slides and incubated
for 20 minutes in A solution (NaCl saturated 80% ethanol with 0.01% NaOH) and were
directly transferred to B solution (solution A saturated with Congo red).
Samples were rinsed in 100% ethanol and xylen and mounted. Amyloid specific
green birefringence was studied in an Olympus X51 microscope (Olympus, Tokyo, Japan)
with two polarising filters connected to an Olympus DP 50 digital camera run by
Studio Lite v 1.0.1 software.


## 2.10. Electron Microscopy

Droplets of sample were placed on formvar coated
copper grids and were negative contrasted with 2% uranyl acetate in 50% ethanol.
Samples were studied in a Jeol 1230
electron microscope at 100 kV (Jeol, Akishima, Tokyo, Japan).
Digital electron micrographs were taken with a Gatan multiscan camera model 791
with corresponding software v3.6.4 (Gatan Inc, Pleasanton, Calif, USA).


## 2.11. Thioflavin Tassay

Kinetic
studies of amyloid formation were performed in Sigmacote (Sigma-Aldrich, St.
Louis, Mo, USA) treated black 96 well plates (Labsystems) in a sample volume of
100μL. Synthetic IAPP was diluted from a DMSO stock
solution (5 mM) to a final concentration of 50 μM in thioflavin T
(ThT) in assay buffer (50 mM glycine, 25 mM sodium phosphate buffer, pH 7.0, and 10 μM ThT). Mature IAPP fibrils
(30 nM) were included
to decrease the amyloid lag phase. Samples consisting of ThT assay buffer with
1% DMSO and ThT assay buffer with 30 nM IAPP fibrils and 1% DMSO were included
in the assay. Fluorescence was measured every thirty minutes at an excitation
wavelength of 442 nm and an emission wavelength of 486 nm. Each sample was
measured in 12 individual wells, and data presented are mean values ±SEM.


## 2.12. Statistical Analysis

Statistics
were performed in GraphPad InStat version 3.06 (GraphPad Software Inc., San Diego, Calif,
USA). Unpairedt-test
was used when two groups were compared and one-way analysis of variance (ANOVA)
with Dunnett multiple comparisons test when several groups were analysed. A
P-value
less than 0.05 was considered significant.


## 3. Results

### 3.1. Evaluation of the Method

Autofluorescence from different solutions was analysed at emission wavelengths 535 nm and 480 nm
(see
Figure1(a)). The KRHG buffer had the lowest fluorescence of the
investigated solutions and was therefore used as assay buffer throughout the
study.Determination
of assay buffer and investigation of pFRET2-DEVD expressing B-TC-6 cells.
(a) Solutions
(KRHG, HBSS, RPMI without FBS, RPMI, Dulbecco´s modified medium, Schneider´s
drosophila medium, and H2O) were
analysed for autofluorescence at wavelength 535 nm (blue columns) and 480 nm (red
columns). Data presented are mean values ±SEM (n=8).
(b) Fluorescence at 535 nm and 480 nm was measured for KRHG buffer (black columns),
B-TC-6 cells expressing pFRET2-DEVD (green columns), and untransfected B-TC-6 cells
(grey columns). Data presented are mean values ±SEM (n=8). Difference in fluorescence signal between B-TC-6
cells expressing pFRET2-DEVD and untransfected B-TC-6 was considered to be
significant (P<.0005) in a two-tailed unpaired t-test.
(c) Confocal image of pFRET2-DEVD
expressing B-TC-6 cells and (d) untransfected
B-TC-6 cells. Argon 488 nm and HeNe 633 nm lasers were used at the same energy levels in
the two images. Green fluorescence indicates expression of pFRET2-DEVD and blue fluorescence nuclear staining TO-PRO-3. Scale bars represent 10 μm.
(a)(b)(c)(d)To determine if the fluorescent signal from the
pFRET2-DEVD transfected B-TC-6 cells was sufficient for detection in the plate
reader the signal was compared to the signal from untransfected B-TC-6 cells. B-TC-6 cells expressing pFRET2-DEVD
had a 6.5-fold higher fluorescence signal at 535 nm and a 2.3-fold higher
signal at 480 nm when compared to the untransfected B-TC-6 cells (see
Figure1(b)).
Fluorescence form transfected B-TC-6 cells differed significantly at both
wavelengths compared to untransfected B-TC-6 cells in an unpaired t-test
(P<.0005). The 535 nm/480 nm ratio for living pFRET2-DEVD transfected B-TC-6
cells was 2.2. Three hours incubation with the apoptosis inducer staurosporine (2 μM)
decreased the FRET ratio to 1.2. Synthetic human and rat IAPP peptides in
monomeric form, at concentrations 25 and 50 μM, were incubated with pFRET2-DEVD
B-TC-6 cells. Only negligible reduction in FRET signal was detected after 12 hour
incubation, and cell survival was calculated to 96% and 98% after incubation
with rat IAPP and human IAPP, respectively. There was no difference between cells incubated with 25 or 50 μM (data
not shown). In the confocal microscope, the pFRET2-DEVD B-TC-6 cells revealed a
strong cytoplasmic fluorescence at excitation wavelength 488 nm (see Figure 1(c)).
This was not present in untransfected cells (see Figure 1(d)).


### 3.2. Amyloid Formation and Loss of FRET

FRET
reduction as a result of amyloid formation was initially investigated with
synthetic IAPP peptides. The amyloid formation from 50μM monomeric IAPP seeded
with 30 nM IAPP fibrils was monitored in the ThT assay and with the FRET assay in
parallel (see Figure 2). In the ThT assay, amyloid specific fluorescence of
seeded IAPP was detected after a 3-hour lag phase and reached a plateau after 9
hours (see Figure 2(a)). No increase in basal fluorescence was detected for the
sample with ThT buffer alone or ThT buffer with low concentration of premade IAPP
seeds. To further study the amyloid aggregation process, aliquots from the
seeded 50 μM IAPP solution were collected hourly and prepared for electron
microscopy. After 2 hours, small ring shaped structures with an outer diameter
of approximately 15 nm could be detected at high magnification (see Figure 2(b)).
After 4 hours of incubation, very thin thread-like fibrils were
detected (see Figure 2(c)) and after 12 hours of incubation fibrils with a more
mature fibrillar appearance were visible (see Figure 2(d)).Figure 2
Parallel performed ThT assay, electron
microscopy, and FRET assay of synthetic IAPP peptide.
(a) ThT
assay of synthetic IAPP peptide (50μM) seeded with preformed IAPP fibrils
(30 nM)
(), ThT assay buffer seeded with preformed
IAPP fibrils (30 nm) (), and ThT assay
buffer (). Data presented are mean values ±SEM (n=12). Synthetic IAPP peptide
(50 μM) seeded with
preformed IAPP fibrils (30 nM) incubated for (b) 2 hours of 50 μM,
(c) for 4 hours, (d) and for 12 hours; all
samples were negative contrasted. In (b), the higher magnification shows small
prefibrillar ring shaped structures. In (c), thin thread-like protofibrils are
visible and in (d) more mature amyloid-like fibrils are apparent. Large-scale
bars represent 100 nm, and the smaller-scale bar in (b) represents 20 nm. (e) FRET ratio of 535 nm/480 nm  measurements of KRHG buffer with seeds of preformed
IAPP fibrils (30 nM) (), synthetic
IAPP peptide (50 μM) seeded with preformed IAPP fibrils
(30 nM) (), and staurosporine
(2 μM)
(). Measurements were performed during a 12-hour
period with individual measurements each hour. Data presented are mean values ±SEM (n=9).
(f) FRET ratio after 12 hours of measurements. Staurosporine sample was set as zero
and was subtracted from assay measurements, and the negative control was set as
100% viable cells (blue column). The FRET ratio of the B-TC-6 cells incubated
in synthetic IAPP peptide (50 μM) seeded with preformed IAPP fibrils
(30 nM) was
37% of the negative control corresponding to a population of 65% apoptotic
cells (green column). Data presented are mean values ±SEM (n=9). The difference between the negative control
and IAPP was considered significant in a two-tailed unpaired t-test (P<.0006).In the FRET measurements, pFRET2-DEVD expressing B-TC-6
cells incubated with staurosporine used as positive control showed an instant reduction
of the 535 nm/480 nm ratio. After 4 hours, the ratio was reduced to 1.2 (see
Figure2(e)). The FRET ratio for pFRET2-DEVD expressing B-TC-6 cells incubated with KRHG
buffer and 30 nM IAPP seeds used as negative control were relative stable at a
ratio of 2.0 through out the assay. pFRET2-DEVD expressing B-TC-6 cells incubated
in 50 μM IAPP peptide with 30 nM IAPP seeds showed a reduced
FRET signal compared to the negative control (see Figure 2(e)).  The
measurements were done hourly for 12 hours, and the calculation was performed
as follows. The FRET ratio 535 nm/480 nm, determined after 12 hours of incubation with the
apoptosis-inducer staurosporine, was set as baseline and corresponds to 0%
survival cells. All cells were considered viable after incubation with preformed
fibrils, and the value for the FRET ratio 535 nm/480 nm was set as 100% after subtraction
of the baseline value.The FRET ratio of the B-TC-6 cells incubated in
amyloid forming IAPP peptide corresponds to a population of 65% apoptotic cells
(see Figure2(f)). B-TC-6 cells incubated in amyloid forming IAPP differed
significantly from the negative control in a two-tailed unpaired t-test
(P<.006).


### 3.3. Characterisation of Recombinant Peptides

The recombinant
peptidesrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C were expressed as fusion proteins and when the
GST-tag was enzymatically removed, all four peptides formed aggregates during the
12 hours time period. The aggregates from the different recombinant peptides stained
with Congo red and revealed green birefringence (see Figures
3(a)–3(d)). Negative
contrasted aggregates showed typical amyloid-like fibrils with a diameter of
10 nm and of variable length (see Figures
3(e)–3(h)).Congo red staining and electron
micrographs of amyloid-like fibrils fromrecproIAPP,
recN+IAPP, recIAPP,
and recIAPP+C.
Congo red
staining viewed in polarised light showing amyloid specific green birefringence
of (a) recproIAPP, (c)  recN+IAPP, (e)
recIAPP, and (g)
recIAPP+C.
Scale bars represent 20 μm. Electron micrographs of negative contrasted
amyloid-like fibrils of (b)  recproIAPP, (d)
recN+IAPP, (f)
recIAPP,
and (h)  recIAPP+C. Scale bars represent 100 nm.
(a)(b)(c)(d)(e)(f)(g)(h)The expected amino acid sequences ofrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C was used to determine the
expected molecular masses (see Figure 4(a)).The produced peptides were run on
Tricine SDS-PAGE and bands corresponding to the molecular masses of each
individual peptide were cut out, trypsinised and analysed by electrospray ionisation tandem mass spectrometry (see Figure
4(b)). The expected tryptic fragments
from each peptide were identified in full scan spectrum (data not shown), and the
sequence of each fragment was confirmed by collision-induced fragmentation mass
spectrometry. All four IAPP peptides had a 10-amino acid residue sequence
consisting of
residues GSRRASVGSP N-terminally
which is a rest from the GST cleavage site. These extra residues did not interfere
with the capability of the recombinant peptides to form amyloid-like fibrils as
shown in Figure 3.Characterisation ofrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C.
(a) A cartoon showing the amino acid sequences of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C peptides. N-terminally, a fragment consisting of 10
residues originating from the GST-tag is present (white box). (b) Silver stained tricine SDS-PAGE of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C. Bands were removed from the tricine
gel, trypsinised and analysed by electrospray
ionisation tandem mass spectrometry.
(a)(b)


### 3.4. Apoptotic Effect of Recombinant Peptides

pFRET2-DEVD
expressing B-TC-6 cells were incubated
with 50μM of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C  together with 30 nM IAPP seeds in KRHG buffer. A
reduction of the FRET ratio was detected during the time laps of the assay (see
Figure 5(a)). After 12 hours of incubation, all four recombinant peptides had
generated a large reduction of the FRET signal. The FRET
ratio of the positive control was set as zero and subtracted from all other
measurements and the negative control was set as 100% viable cells. The FRET ratios
for recproIAPP, recN+IAPP, recIAPP, and recIAPP+C were 51%, 54%, 28%, and 41%,
respectively, when compared to the negative control. This corresponds to a
population of 49%, 46%, 72%, and 59% apoptotic cells (see Figure 5(b)). All
four groups differed significantly from the negative control in an ANOVA test (P < .0005).
No significant difference within the four recombinant peptide groups was detected
in an ANOVA test (P>.2).FRET assays ofrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C. (a) B-TC-6 cells expressing pFRET2-DEVD were incubated with solubilised
recombinant peptides recproIAPP (50 μM)
(), recN+IAPP
(50 μM)
(), recIAPP (50 μM)
(), and recIAPP+C (50 μM)
(), all
supplemented with preformed IAPP fibrils at a concentration of 30 nM or
incubated with preformed IAPP fibrils (30 nM) alone (),
or staurosporine (2 μM)
(). The FRET ratio
at 535/480 nm was determined hourly over 12 hours. Data presented are mean value
±SEM (n=5). (b) Cell viability after incubation
for 12 hours with solubilised recombinant peptides supplemented with preformed
IAPP fibrils (30 nM). Data are presented as mean value ±SEM (n=5). Relative to the control, cell viability was
determined to 51% for recproIAPP (), 54% for recN+IAPP
(), 28% for
recIAPP (),
and 41% for recIAPP+C ().
Compared to the control, a significant difference in the FRET ratio was
observed for all four recombinant peptides in an ANOVA
test (P<.0005). (c) FRET ratio of pFRET2-DEVD
expressing B-TC-6 cells after 12 hours of incubation with recombinant
amyloid-like fibrils corresponding to 50 μM. RecproIAPP
(), recN+IAPP
(), recIAPP
(), and recIAPP+C
() and control incubation with buffer alone
().
Relative to the control, cell viability after incubation with recproIAPP was 85%, with recN+IAPP 102%, with recIAPP 104% and with recIAPP+C 89%. No statistically
difference between the negative control and the four recombinant peptides was
observed in an ANOVA test (P<.5). (d) Incubation of pFRET2-DEVD expressing B-TC-6 cells with recombinant
peptides in solubilised form (50 μM). RecproIAPP
(), recN+IAPP
(), recIAPP
(), and recIAPP+C
() and
control incubation with buffer alone ().  Relative to the control, cell viability after
12 hours incubation with recproIAPP
was 86%, recN+IAPP 97%, recIAPP 102%, and recIAPP+C 89%. The four recombinant peptides did not differ significantly
from the negative control in an ANOVA test (P>.06).
(a)(b)(c)(d)pFRET2-DEVD expressing B-TC-6 cells was also incubated with
mature amyloid-like fibrils fromrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C. No
difference in FRET ratio was observed over time when compared to the negative
control (data not shown). After 12 hours of incubation,   FRET ratios of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C were 85%, 102%, 104%, and 89%,
respectively, when the positive control was subtracted and the negative control
was set as 100% viable cells (see Figure 5(c)). No significant differences
between the negative control and the recombinant fibrils and no difference
between the four different peptides (P>.5) were detected.FRET measurements were also performed on pFRET2-DEVD
expressing B-TC-6 cells incubated with 50μM solubilised and filtered recombinant
peptides. When compared to the negative control, no loss of FRET was detected
during 12 hours of measurements (data not shown). After 12 hours of incubation
and subtraction of the positive control, FRET ratios for recproIAPP, recN+IAPP, recIAPP, and recIAPP+C were 86%, 97%, 102%, and 89%, respectively, compared to
the negative control (see Figure 5(d)). No significant difference between the negative
control and the monomeric peptides and no differences between the four
different recombinant peptides (P>.06) were detected.


## 3.1. Evaluation of the Method

Autofluorescence from different solutions was analysed at emission wavelengths 535 nm and 480 nm
(see
Figure1(a)). The KRHG buffer had the lowest fluorescence of the
investigated solutions and was therefore used as assay buffer throughout the
study.Determination
of assay buffer and investigation of pFRET2-DEVD expressing B-TC-6 cells.
(a) Solutions
(KRHG, HBSS, RPMI without FBS, RPMI, Dulbecco´s modified medium, Schneider´s
drosophila medium, and H2O) were
analysed for autofluorescence at wavelength 535 nm (blue columns) and 480 nm (red
columns). Data presented are mean values ±SEM (n=8).
(b) Fluorescence at 535 nm and 480 nm was measured for KRHG buffer (black columns),
B-TC-6 cells expressing pFRET2-DEVD (green columns), and untransfected B-TC-6 cells
(grey columns). Data presented are mean values ±SEM (n=8). Difference in fluorescence signal between B-TC-6
cells expressing pFRET2-DEVD and untransfected B-TC-6 was considered to be
significant (P<.0005) in a two-tailed unpaired t-test.
(c) Confocal image of pFRET2-DEVD
expressing B-TC-6 cells and (d) untransfected
B-TC-6 cells. Argon 488 nm and HeNe 633 nm lasers were used at the same energy levels in
the two images. Green fluorescence indicates expression of pFRET2-DEVD and blue fluorescence nuclear staining TO-PRO-3. Scale bars represent 10 μm.
(a)(b)(c)(d)To determine if the fluorescent signal from the
pFRET2-DEVD transfected B-TC-6 cells was sufficient for detection in the plate
reader the signal was compared to the signal from untransfected B-TC-6 cells. B-TC-6 cells expressing pFRET2-DEVD
had a 6.5-fold higher fluorescence signal at 535 nm and a 2.3-fold higher
signal at 480 nm when compared to the untransfected B-TC-6 cells (see
Figure1(b)).
Fluorescence form transfected B-TC-6 cells differed significantly at both
wavelengths compared to untransfected B-TC-6 cells in an unpaired t-test
(P<.0005). The 535 nm/480 nm ratio for living pFRET2-DEVD transfected B-TC-6
cells was 2.2. Three hours incubation with the apoptosis inducer staurosporine (2 μM)
decreased the FRET ratio to 1.2. Synthetic human and rat IAPP peptides in
monomeric form, at concentrations 25 and 50 μM, were incubated with pFRET2-DEVD
B-TC-6 cells. Only negligible reduction in FRET signal was detected after 12 hour
incubation, and cell survival was calculated to 96% and 98% after incubation
with rat IAPP and human IAPP, respectively. There was no difference between cells incubated with 25 or 50 μM (data
not shown). In the confocal microscope, the pFRET2-DEVD B-TC-6 cells revealed a
strong cytoplasmic fluorescence at excitation wavelength 488 nm (see Figure 1(c)).
This was not present in untransfected cells (see Figure 1(d)).


## 3.2. Amyloid Formation and Loss of FRET

FRET
reduction as a result of amyloid formation was initially investigated with
synthetic IAPP peptides. The amyloid formation from 50μM monomeric IAPP seeded
with 30 nM IAPP fibrils was monitored in the ThT assay and with the FRET assay in
parallel (see Figure 2). In the ThT assay, amyloid specific fluorescence of
seeded IAPP was detected after a 3-hour lag phase and reached a plateau after 9
hours (see Figure 2(a)). No increase in basal fluorescence was detected for the
sample with ThT buffer alone or ThT buffer with low concentration of premade IAPP
seeds. To further study the amyloid aggregation process, aliquots from the
seeded 50 μM IAPP solution were collected hourly and prepared for electron
microscopy. After 2 hours, small ring shaped structures with an outer diameter
of approximately 15 nm could be detected at high magnification (see Figure 2(b)).
After 4 hours of incubation, very thin thread-like fibrils were
detected (see Figure 2(c)) and after 12 hours of incubation fibrils with a more
mature fibrillar appearance were visible (see Figure 2(d)).Figure 2
Parallel performed ThT assay, electron
microscopy, and FRET assay of synthetic IAPP peptide.
(a) ThT
assay of synthetic IAPP peptide (50μM) seeded with preformed IAPP fibrils
(30 nM)
(), ThT assay buffer seeded with preformed
IAPP fibrils (30 nm) (), and ThT assay
buffer (). Data presented are mean values ±SEM (n=12). Synthetic IAPP peptide
(50 μM) seeded with
preformed IAPP fibrils (30 nM) incubated for (b) 2 hours of 50 μM,
(c) for 4 hours, (d) and for 12 hours; all
samples were negative contrasted. In (b), the higher magnification shows small
prefibrillar ring shaped structures. In (c), thin thread-like protofibrils are
visible and in (d) more mature amyloid-like fibrils are apparent. Large-scale
bars represent 100 nm, and the smaller-scale bar in (b) represents 20 nm. (e) FRET ratio of 535 nm/480 nm  measurements of KRHG buffer with seeds of preformed
IAPP fibrils (30 nM) (), synthetic
IAPP peptide (50 μM) seeded with preformed IAPP fibrils
(30 nM) (), and staurosporine
(2 μM)
(). Measurements were performed during a 12-hour
period with individual measurements each hour. Data presented are mean values ±SEM (n=9).
(f) FRET ratio after 12 hours of measurements. Staurosporine sample was set as zero
and was subtracted from assay measurements, and the negative control was set as
100% viable cells (blue column). The FRET ratio of the B-TC-6 cells incubated
in synthetic IAPP peptide (50 μM) seeded with preformed IAPP fibrils
(30 nM) was
37% of the negative control corresponding to a population of 65% apoptotic
cells (green column). Data presented are mean values ±SEM (n=9). The difference between the negative control
and IAPP was considered significant in a two-tailed unpaired t-test (P<.0006).In the FRET measurements, pFRET2-DEVD expressing B-TC-6
cells incubated with staurosporine used as positive control showed an instant reduction
of the 535 nm/480 nm ratio. After 4 hours, the ratio was reduced to 1.2 (see
Figure2(e)). The FRET ratio for pFRET2-DEVD expressing B-TC-6 cells incubated with KRHG
buffer and 30 nM IAPP seeds used as negative control were relative stable at a
ratio of 2.0 through out the assay. pFRET2-DEVD expressing B-TC-6 cells incubated
in 50 μM IAPP peptide with 30 nM IAPP seeds showed a reduced
FRET signal compared to the negative control (see Figure 2(e)).  The
measurements were done hourly for 12 hours, and the calculation was performed
as follows. The FRET ratio 535 nm/480 nm, determined after 12 hours of incubation with the
apoptosis-inducer staurosporine, was set as baseline and corresponds to 0%
survival cells. All cells were considered viable after incubation with preformed
fibrils, and the value for the FRET ratio 535 nm/480 nm was set as 100% after subtraction
of the baseline value.The FRET ratio of the B-TC-6 cells incubated in
amyloid forming IAPP peptide corresponds to a population of 65% apoptotic cells
(see Figure2(f)). B-TC-6 cells incubated in amyloid forming IAPP differed
significantly from the negative control in a two-tailed unpaired t-test
(P<.006).


## 3.3. Characterisation of Recombinant Peptides

The recombinant
peptidesrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C were expressed as fusion proteins and when the
GST-tag was enzymatically removed, all four peptides formed aggregates during the
12 hours time period. The aggregates from the different recombinant peptides stained
with Congo red and revealed green birefringence (see Figures
3(a)–3(d)). Negative
contrasted aggregates showed typical amyloid-like fibrils with a diameter of
10 nm and of variable length (see Figures
3(e)–3(h)).Congo red staining and electron
micrographs of amyloid-like fibrils fromrecproIAPP,
recN+IAPP, recIAPP,
and recIAPP+C.
Congo red
staining viewed in polarised light showing amyloid specific green birefringence
of (a) recproIAPP, (c)  recN+IAPP, (e)
recIAPP, and (g)
recIAPP+C.
Scale bars represent 20 μm. Electron micrographs of negative contrasted
amyloid-like fibrils of (b)  recproIAPP, (d)
recN+IAPP, (f)
recIAPP,
and (h)  recIAPP+C. Scale bars represent 100 nm.
(a)(b)(c)(d)(e)(f)(g)(h)The expected amino acid sequences ofrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C was used to determine the
expected molecular masses (see Figure 4(a)).The produced peptides were run on
Tricine SDS-PAGE and bands corresponding to the molecular masses of each
individual peptide were cut out, trypsinised and analysed by electrospray ionisation tandem mass spectrometry (see Figure
4(b)). The expected tryptic fragments
from each peptide were identified in full scan spectrum (data not shown), and the
sequence of each fragment was confirmed by collision-induced fragmentation mass
spectrometry. All four IAPP peptides had a 10-amino acid residue sequence
consisting of
residues GSRRASVGSP N-terminally
which is a rest from the GST cleavage site. These extra residues did not interfere
with the capability of the recombinant peptides to form amyloid-like fibrils as
shown in Figure 3.Characterisation ofrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C.
(a) A cartoon showing the amino acid sequences of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C peptides. N-terminally, a fragment consisting of 10
residues originating from the GST-tag is present (white box). (b) Silver stained tricine SDS-PAGE of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C. Bands were removed from the tricine
gel, trypsinised and analysed by electrospray
ionisation tandem mass spectrometry.
(a)(b)


## 3.4. Apoptotic Effect of Recombinant Peptides

pFRET2-DEVD
expressing B-TC-6 cells were incubated
with 50μM of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C  together with 30 nM IAPP seeds in KRHG buffer. A
reduction of the FRET ratio was detected during the time laps of the assay (see
Figure 5(a)). After 12 hours of incubation, all four recombinant peptides had
generated a large reduction of the FRET signal. The FRET
ratio of the positive control was set as zero and subtracted from all other
measurements and the negative control was set as 100% viable cells. The FRET ratios
for recproIAPP, recN+IAPP, recIAPP, and recIAPP+C were 51%, 54%, 28%, and 41%,
respectively, when compared to the negative control. This corresponds to a
population of 49%, 46%, 72%, and 59% apoptotic cells (see Figure 5(b)). All
four groups differed significantly from the negative control in an ANOVA test (P < .0005).
No significant difference within the four recombinant peptide groups was detected
in an ANOVA test (P>.2).FRET assays ofrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C. (a) B-TC-6 cells expressing pFRET2-DEVD were incubated with solubilised
recombinant peptides recproIAPP (50 μM)
(), recN+IAPP
(50 μM)
(), recIAPP (50 μM)
(), and recIAPP+C (50 μM)
(), all
supplemented with preformed IAPP fibrils at a concentration of 30 nM or
incubated with preformed IAPP fibrils (30 nM) alone (),
or staurosporine (2 μM)
(). The FRET ratio
at 535/480 nm was determined hourly over 12 hours. Data presented are mean value
±SEM (n=5). (b) Cell viability after incubation
for 12 hours with solubilised recombinant peptides supplemented with preformed
IAPP fibrils (30 nM). Data are presented as mean value ±SEM (n=5). Relative to the control, cell viability was
determined to 51% for recproIAPP (), 54% for recN+IAPP
(), 28% for
recIAPP (),
and 41% for recIAPP+C ().
Compared to the control, a significant difference in the FRET ratio was
observed for all four recombinant peptides in an ANOVA
test (P<.0005). (c) FRET ratio of pFRET2-DEVD
expressing B-TC-6 cells after 12 hours of incubation with recombinant
amyloid-like fibrils corresponding to 50 μM. RecproIAPP
(), recN+IAPP
(), recIAPP
(), and recIAPP+C
() and control incubation with buffer alone
().
Relative to the control, cell viability after incubation with recproIAPP was 85%, with recN+IAPP 102%, with recIAPP 104% and with recIAPP+C 89%. No statistically
difference between the negative control and the four recombinant peptides was
observed in an ANOVA test (P<.5). (d) Incubation of pFRET2-DEVD expressing B-TC-6 cells with recombinant
peptides in solubilised form (50 μM). RecproIAPP
(), recN+IAPP
(), recIAPP
(), and recIAPP+C
() and
control incubation with buffer alone ().  Relative to the control, cell viability after
12 hours incubation with recproIAPP
was 86%, recN+IAPP 97%, recIAPP 102%, and recIAPP+C 89%. The four recombinant peptides did not differ significantly
from the negative control in an ANOVA test (P>.06).
(a)(b)(c)(d)pFRET2-DEVD expressing B-TC-6 cells was also incubated with
mature amyloid-like fibrils fromrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C. No
difference in FRET ratio was observed over time when compared to the negative
control (data not shown). After 12 hours of incubation,   FRET ratios of recproIAPP, recN+IAPP, recIAPP, and recIAPP+C were 85%, 102%, 104%, and 89%,
respectively, when the positive control was subtracted and the negative control
was set as 100% viable cells (see Figure 5(c)). No significant differences
between the negative control and the recombinant fibrils and no difference
between the four different peptides (P>.5) were detected.FRET measurements were also performed on pFRET2-DEVD
expressing B-TC-6 cells incubated with 50μM solubilised and filtered recombinant
peptides. When compared to the negative control, no loss of FRET was detected
during 12 hours of measurements (data not shown). After 12 hours of incubation
and subtraction of the positive control, FRET ratios for recproIAPP, recN+IAPP, recIAPP, and recIAPP+C were 86%, 97%, 102%, and 89%, respectively, compared to
the negative control (see Figure 5(d)). No significant difference between the negative
control and the monomeric peptides and no differences between the four
different recombinant peptides (P>.06) were detected.


## 4. Discussion

The
pFRET2-DEVD vector has earlier been demonstrated by Köhler et al. to be an excellent reporter for apoptosis induced by caspase-3-like
proteases in the insulin producing beta-cell lines RINm5F and MIN6 [29]. By measuring loss of FRET on individual transiently transfected cells,
the course of apoptosis was monitored in real-time. Here, we used the pFRET2-DEVD vector to establish a novel assay
with stable transfected B-TC-6 cells that allows accurate real-time studies of
apoptosis in 96 well plates. The assay was used for studies of beta-cell
apoptosis during amyloid formation, and the toxicity of recproIAPP and the processing metabolites recN+IAPP, recIAPP, and recIAPP+C is evaluated.Initially,
it was essential to find an assay
buffer with very low autofluorescence for measurement of FRET signal from cells
cultured in a black 96 well plate. The KRHG-buffer revealed the lowest
background and was selected for the assay, but with the disadvantage of being a
rather poor medium. After 12 hours of FRET measurement, the negative control
started to show some reduction of FRET and therefore measurements beyond this
time point are not presented throughout this work. The fluorescent signal detected
from the pFRET2-DEVD transfected B-TC-6 cells was adequate for plate reader
measurements and since it is the ratio between 535 and 480 nm, that is, given differences
in cell density can be ignored. Synthetic IAPP peptide was initially used for the
setup of the method.One
step in the evaluation procedure of the FRET method was to determine the
amyloid fibril formation progress under the used peptide concentrations and
correlate it to the induction of apoptosis. For this, a ThT assay was run in
parallel with identical IAPP concentrations. When monomeric IAPP was seeded
with preformed IAPP amyloid-like fibrils, an increase in ThT-fluorescence signal
appeared after a three hours lag phase and reached maximum after nine hours of
incubation. Fibril formation could only be detected in seeded IAPP samples, and
not in samples with seed alone. In samples collected for morphological
evaluation, an amorphous material was present after two hours and at higher
resolution ring-like structures with an outer diameter of 15 nm could be seen.
After four hours of incubation, thin slender fibrils were present. Since no
fibrilar material was present at the earlier time points we conclude that these
thin fibrils were newly formed and did not represent the low amount of
sonicated preformed fibrils added as seed. In samples taken after 12 hours,
fibrils with a more distinct fibrilar appearance had emerged. The decrease in
FRET ratio after incubation with 2μM staurosporine starts under the first hour
and continues over a five-hour period. Staurosporine is a potent inducer of
apoptosis, a transient biological process that persists for 3-4 hours. In this
positive control, all cells undergo apoptosis. In transfected cells incubated
with monomeric IAPP and preformed fibrils, apoptosis was initiated after
approximately seven hours and this is in line with the expected time frame if
oligomers are responsible for the induction of apoptosis.The B-TC-6 cell line is of mouse origin, and it has previously been suggested
that mouse IAPP has an antiamyloidogenic effect on human IAPP at least intracellularly
[31]. In vitro studies show that insulin also has an
inhibitory property on amyloid formation of human IAPP [32, 33]. Prior to the experimental procedure, the B-TC-6 cells were washed in
KRHG buffer which has a low glucose concentration (2 mM). Therefore, exocytosis
of mouse IAPP and insulin was considered to be very low during the assay and not
to influence the amyloidogenicity of human IAPP.The predominant hypothesis
of amyloid cytotoxicity is that the prefibrillar assemblies are able to
incorporate into cellular membranes causing cation influx [10, 34, 35]. Oligomeric species from different peptides would
there share a common structure and act cytotoxic by the same mechanisms. A
common pathway for apoptosis triggered by amyloid pore formation has not yet
been proposed. Apoptosis is the major cause of beta-cell reduction in type 2
diabetic patients and has been shown to occur during formation of islet amyloid
in a transgenic mouse model [15, 36]. Two apoptosis studies on beta-cell lines have been performed where activation
of the caspase cascade in parallel to IAPP amyloid formation has been observed [19, 20]. Both authors have used RINm5F cells and Zhang el al. have also used the human insulinoma cell line CM. Both
groups reported the activation of the common downstream protease caspase 3 with
upstream activated JNK pathway (c-jun NH2-terminal kinase/stress-activated
protein kinase). Zhang et al. also reported activated caspase 8 and
caspase 1 proteases upstream of caspase 3. The exact pathways of IAPP triggered
apoptosis are not clear but caspase 3 acts as a common downstream effector. Zhang et al. state that caspase 3
activation was detected after 8 hours of incubation with preseeded synthetic IAPP
and reached a maximum after 16 hours [20]. No measurements between these time points were performed. Our
observation of the reduced FRET signal goes well with these data, and upstream
activation must occur before we detect FRET reduction by caspase 3 activation.All four
recombinant peptides,recproIAPP, recN+IAPP, recIAPP, and recIAPP+C
possess amyloidogenic properties and form fibrillar aggregates. The extra amino
acid residues N-terminally of the recombinant peptides do not interfere with
the amyloidogenic propensity since all four peptides form amyloid-like fibrils.
The apoptotic effects of the recombinant peptides were investigated using the developed
FRET assay. The recombinant peptides were studied in solubilised seeded, fibrillar,
and in solubilised nonseeded form. A loss of FRET was detected for all four
seeded solubilised recombinant peptides. At the end of the assay, an apoptotic
rate ranging form 46–72% was observed for the four different seeded recombinant
peptides though no statistically significant difference between the groups was distinguished.
Important, the degree of apoptosis did not differ between cells incubated with synthetic
IAPP (65%) or recIAPP (72%) (P>.5)
pointing out that the addition of the 10 residues at the N-terminus of the
recombinant IAPP did not interfere with the cytotoxicity of the peptide.B-TC-6 cells expressing pFRET2-DEVD
were incubated with mature amyloid fibrils fromrecproIAPP, recN+IAPP, recIAPP, and recIAPP+C and no apoptosis was detected. Since the amyloid-like fibrils
were extensively washed with water, a pure fibril fraction without any
oligomeric species would be expected. Solubilised nonseeded recombinant peptides
did not elicit a reduction of the FRET signal, most likely due to a long lag
phase. From this we conclude that the cytotoxic species of synthetic and
recombinant amyloidogenic IAPP peptides are early oligomers or protofibrils.
This is in consensus with earlier studies [16, 17, 37].A new and very interesting
finding is thatrecproIAPP and the
processing intermediates recN+IAPP
and recIAPP+C trigger beta-cell
apoptosis. Earlier we and others have shown that islet amyloid deposition may
start intracellularly and that aberrant processing of proIAPP can be an
initiating event [26, 27]. Intracellular deposits containing proIAPP have been found both in
transgenic mice expressing human IAPP and in human beta-cells transplanted
under the renal capsule of mice fed a high fat diet [28]. In the latter study, we also found intra-granular amyloid-like fibrils
consisting of proIAPP. It has previously been shown that amyloid formation of
IAPP was strongly enhanced by phospholipid bilayers and that amyloid elongation
occurred from the surface of the lipid bilayer [38]. Hypothetically, the membrane of the secretory granule or endoplasmatic
reticulum may be the location of initial amyloid formation and where oligomeric
pore structures can incorporate. By exocytosis, the granule membrane will fuse
with the outer cell membrane and a cation influx and activation of apoptotic
pathways might occur. Here, we demonstrate that recproIAPP and the processing intermediates recN+IAPP and recIAPP+C can
induce caspase 3 activation and trigger beta-cell apoptosis. This is achieved
by prefibrillar toxic species since fibrillar and monomeric forms of the
recombinant peptides did not trigger caspase 3-like activity. This further
strengthens our hypothesis that aberrant processing of proIAPP plays a key role
in early islet amyloidogenesis.Apoptosis is a transient
event, and the established assay allows analysis over time of the same cell
population. It is a system with high reproducibility, and it is cost effective
and easy to handle. Apoptosis assays where Ac-DEVD-AMC is used as fluorogenic
substrate is often labour intense and the commonly used MTT assay, where only
living cells are measured, is not a true assay for apoptosis. The Vybrant
apoptosis detection kit uses three different fluorescent nuclear stains with the propensity to penetrate cell
membrane according to an apoptotic or nonapoptotic state. This method is
performed on living cells and must be analysed microscopically. It is work intense
and dependent on the interpretation of the analyst. Also TUNEL and DNA
fragmentation methods are labour intense and similar to all the methods
described above, only measurement at one time point is possible. Factors such
as equal amounts of cells in each well or assay and rate of cell division are
very important to consider when to deal with the described methods. The
weakness of our FRET assay is the poor medium in which the assay is performed.
Further investigations of possible media to prolong the survival of the cells
and thereby allow measurements over a more extended period of time would be
valuable. Future prospects for this method are to study apoptosis in presence
of antiamyloidogenic or amyloid enhancing factors depending on strategy to
reduce toxic species in the amyloid forming process.



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*Source: 865850-2008-06-10.xml*