Designed peptides as nanomolar crossamyloid inhibitors acting via supramolecular nanofiber co-assembly

3.0 科研～小助 2025-09-01 4 4 8.59MB 22 页 1知币
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nature communications
Article https://doi.org/10.1038/s41467-022-32688-0
Designed peptides as nanomolar cross-
amyloid inhibitors acting via supramolecular
nanofiber co-assembly
Karin Taş
1
, Beatrice Dalla Volta
1
, Christina Lindner
1,10
, Omar El Bounkari
2
,
Kathleen Hille
1
,YuanTian
2
, Xènia Puig-Bosch
3
, Markus Ballmann
3
,
Simon Hornung
1
, Martin Ortner
1,11
, Sophia Prem
1,12
,LauraMeier
4
,
Gerhard Rammes
3
,MartinHaslbeck
4
, Christian Weber
5,6,7,8
,
RemcoT.A.Megens
5,7,9
, Jürgen Bernhagen
2,6
& Aphrodite Kapurniotu
1
Amyloid self-assembly is linked to numerous devastating cell-degenerative
diseases. However, designing inhibitors of this pathogenic process remains a
major challenge. Cross-interactions between amyloid-βpeptide (Aβ)andislet
amyloid polypeptide (IAPP), key polypeptides of Alzheimer’sdisease(AD)and
type 2 diabetes (T2D), have been suggested to link AD with T2D pathogenesis.
Here, we show that constrained peptides designed to mimic the Aβamyloid
core (ACMs) are nanomolar cross-amyloid inhibitors of both IAPP and Aβ42
and effectively suppress reciprocal cross-seeding. Remarkably, ACMs act by
co-assembling with IAPP or Aβ42 into amyloid fibril-resembling but non-toxic
nanofibers and their highly ordered superstructures. Co-assembled nanofibers
exhibit various potentially beneficial features including thermolability, pro-
teolytic degradability, and effective cellular clearance which are reminiscent of
labile/reversible functional amyloids. ACMs are thus promising leads for
potent anti-amyloid drugs in both T2D and AD while the supramolecular
nanofiber co-assemblies should inform the design of novel functional (hetero-)
amyloid-based nanomaterials for biomedical/biotechnological applications.
Amyloid self-assembly is linked to numerous devastating cell-
degenerative diseases, with AD and T2D being two of the most
prominent ones1,2. The main component of amyloid plaques in AD
brainsisthe40(42)-residuepeptideAβ40(42), while pancreatic
amyloid of T2D patients consists of fibrillar assemblies of the 37-
residue IAPP2,3(Fig. 1a). IAPP is secreted from pancreatic β-cells
and functions as a neuroendocrine regulator of glucose
homeostasis3. However, the formation of cytotoxic IAPP
Received: 16 January 2022
Accepted: 10 August 2022
Check for updates
1
Division of Peptide Biochemistry, TUM School of Life Sciences, Technical University of Munich (TUM), 85354 Freising, Germany.
2
Division of Vascular Biology,
Institute for Stroke and Dementia Research (ISD), Klinikum der Universität München, Ludwig-Maximilian-University (LMU), 81377 Munich, Germany.
3
Department of Anesthesiology and Intensive Care, Technical University of Munich/Klinikum Rechts der Isar, 81675 München, Germany.
4
Center for Protein
Assemblies, Department of Chemistry, Technical University of Munich, 85748 Garching, Germany.
5
Institute for Cardiovascular Prevention, Klinikum der
Universität München, Ludwig-Maximilian-University Munich (LMU), 80336 Munich, Germany.
6
Munich Cluster for Systems Neurology (SyNergy), 81377
Munich, Germany.
7
German Centre for Cardiovascular Research (DZHK), partner site Munich Heart Alliance, 80802 Munich, Germany.
8
Department of
Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 Maastricht, The Netherlands.
9
Department of Biomedical
Engineering, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, 6229 Maastricht, The Netherlands.
10
Present address: Institute of
Organic Chemistry, Centre for Advanced Materials, 69120 Heidelberg, Germany.
11
Present address: Chair of Biopolymer Chemistry, TUM School of Life
Sciences, Technical University of Munich (TUM), 85354 Freising, Germany.
12
Present address: Werner Siemens-Chair of Synthetic Biotechnology (WSSB),
Technical University of Munich (TUM), Department of Chemistry, 85748 Garching, Germany. e-mail: akapurniotu@mytum.de
Nature Communications | (2022) 13:5004 1
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0 12243648
0
10000
20000
30000
40000
50000
60000
ThT fluorescence (arb. units)
Time (h)
IAPP
+ fIAPP (10%)
+ Nle3-VF + fIAPP (10%)
+ L3-VF + fIAPP (10%)
+ F3-VF + fIAPP (10%)
0 12243648
0
10000
20000
30000
70000
80000
90000
100000
+ L3-VF + fAE42 (10%)
+ F3-VF + fAE42 (10%)
+ F3-LF + fAE42 (10%)
ThT fluorescence (arb. units)
Time (h)
IAPP
+ fAE42 (10%)
+ Nle3-VF + fAE42 (10%)
200 210 220 230 240 250
-10000
-5000
0
5000
10000
15000
20000
[
T
T
]
(
deg.cm
2
.dmol
-1
)
Wavelength (nm)
Nle3-LF
L3-LF
F3-LF
VGS-LF
-10-9-8-7-6
0
40
50
60
70
80
90
100
110
MTT reduction (% of control)
Log(IAPP concentration) (M)
IAPP
IAPP + Nle3-LF
IAPP + L3-LF
IAPP + F3-LF
IAPP + VGS-LF
200 210 220 230 240 250
-10000
-5000
0
5000
10000
15000
20000
[
T
]
(
deg.cm
2
.dmol
-1
)
Wavelength (nm)
Nle3-VF
L3-VF
F3-VF
VGS-VF
gi
k
j
d
b
e
h
-10 -9 -8 -7 -6
0
40
50
60
70
80
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100
110
MTT reduction (% of control)
Log(IAPP concentration) (M)
IAPP
IAPP + Nle3-VF
IAPP + L3-VF
IAPP + F3-VF
IAPP + VGS-VF
0 24 48 72 96 120 144 168
0
50
100
150
200
250
ThT fluorescence (arb. units)
Time (h)
IAPP + F3-LF
IAPP + VGS-LF
IAPP
IAPP + Nle3-LF
IAPP + L3-LF
0 24 48 72 96 120 144 168
0
50
100
150
200
250
ThT fluorescence (arb. units)
Time (h)
IAPP + F3-VF
IAPP + VGS-VF
IAPP
IAPP + Nle3-VF
IAPP + L3-VF
a
f
c
Fig. 1 | ACM design concept, their effects on IAPP amyloid self-assembly and
cytotoxicity, and ACM secondary structures. a Sequences of IAPP and Aβ40(42),
proposed models of fIAPP and fAβ40 folds, and hypothetical IAPP/Aβ40 “hetero-
amyloids”(β-strands, pink or blue and underlined; “hot segments”of self-/cross-
interactions, bold; loop residues, italics)25,30,31.bACM inhibitor design strategy.
Template Aβ(15–40) in a β-strand-loop-β-strand fold proposed for fAβ4031 is
modified via (a)N-methylations in Aβ(17–20), bsubstitution of Aβ(24-26) by
hydrophobic tripeptides, and cMet35 substitution by Nle. cSequences of the six
ACMs and negative controls VGS-VF and VGS-LF (Supplementary Table 1). Each
sequence corresponds to two different ACMs which contain the same LTS but a
different couple of N-methylated residues (dashed boxes). Color code as in a;LTS
and tripeptide VGS in red; green or violet for peptide names and corresponding
N-methylated residues. dNle3-VF, L3-VF, and F3-VF block IAPP amyloid self-
assembly. Fibrillogenesis of IAPP (16.5 µM) alone or with ACMs or VGS-VF was
assessed via ThT binding (IAPP/peptide 1/2) (means ± SD, n= 3 independent
assays). eNle3-VF, L3-VF, and F3-VF suppress the formation of toxic IAPP
assemblies. Solutions of d(7-day-aged (VFS-VF 24 h)) added to RIN5fm cells; cell
viability determined via MTT reduction (means ± SD, three independent assays,
n= 3 technical replicates each). fNle3-LF, L3-LF, and F3-LF block IAPP amyloid self-
assembly. Assay as in d(IAPP/peptide 1/2 except L3-LF (1/2.5)) (means ± SD, three
independent assays). gNle3-LF, L3-LF, and F3-LF suppress the formation of toxic
IAPP assemblies. Solutions of f(7-day-aged (VGS-LF 24 h)) added to RIN5fm cells;
cell viability determined via MTT reduction (means ± SD, three independent assays,
n= 3 technical replicates each). h,iSecondary structure of ACMs. Far-UV CD
spectra of ACMs of dand fversus non-inhibitors (5 µM, pH 7.4). jACMs inhibit
seeding of IAPP by preformed fIAPP. Fibrillogenesis of IAPP (12 µM) without or with
fIAPP seeds (10%) and seeded IAPP/ACM mixtures assessed via ThT binding (IAPP/
ACM 1/2) (means ± SD, n= 9 (for IAPP alone) or 3 (for all other samples) indepen-
dent assays). kACMs inhibit fAβ42-mediated cross-seeding of IAPP. Fibrillogenesis
of IAPP with and without fAβ42 seeds (10%) versus IAPP/ACM mixtures (IAPP 12 µM,
IAPP/ACM 1/2) (means ± SD, n= 6 (for IAPP with or without seeds) or n= 3 (for all
other samples) independent assays).
Article https://doi.org/10.1038/s41467-022-32688-0
Nature Communications | (2022) 13:5004 2
Designed peptides as nanomolar crossamyloid inhibitors acting via supramolecular nanofiber co-assembly.pdf

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