FTIR reveals structural differences between native β-sheet proteins and amyloid fibrils

Giorgia Zandomeneghi; Mark R.H. Krebs; Margaret G. McCammon; Marcus Fändrich

doi:10.1110/ps.041024904

Exploration of insulin amyloid polymorphism using Raman spectroscopy and imaging

10.1101/782672 ◽

2019 ◽

Author(s):

M. Ishigaki ◽

K. Morimoto ◽

E. Chatani ◽

Y. Ozaki

Keyword(s):

Disulfide Bond ◽

Amyloid Fibrils ◽

Amyloid Fibril ◽

Relative Proportion ◽

Raman Imaging ◽

Hydroxyl Groups ◽

Structural Differences ◽

Α Helix ◽

Β Sheet

AbstractWe aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature, and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90 ℃. The intensity ratio of two Raman bands at 1672 and 1657 cm-1 due to β-sheet and α-helix structures was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the β-sheet structure was higher in the case with salts, especially at a higher temperature and with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm-1) and the ratio of the tyrosine doublet R(I850⁄I826) were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis (PCA). Especially, amyloid fibrils with Na2SO4 media formed the g-g-g conformation of disulfide bond at a higher rate and without any salts; on the contrary, the g-g-g conformation was partially transformed into the g-g-t conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three-dimensional structural differences of amyloid polymorphisms. The present results indicate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.Statement of SignificanceOur results revealed three Raman markers distinguishing amyloid fibril polymorphisms caused by salt and temperature effects; the relative proportion of protein secondary structures (α–helix and β-sheet), the ratio of tyrosine doublet, and the conformational differences of disulfide bonds. The lower values of tyrosine doublet in the case with salts were interpreted as the anions rob the hydration water from proteins which induced protein misfolding. Using these parameters, Raman images captured their higher order structural differences in situ without labeling. The images of hydrogen bonds strength variations due to tyrosine doublet is believed to include significant novelty. The present results imply the potential of Raman imaging for use as a diagnostic imaging tool for tissues with amyloid-induced diseases.

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Pancreatic beta-cell granule peptides form heteromolecular complexes which inhibit islet amyloid polypeptide fibril formation

Biochemical Journal ◽

10.1042/bj20030852 ◽

2004 ◽

Vol 377 (3) ◽

pp. 709-716 ◽

Cited By ~ 67

Author(s):

Emma T. A. S. JAIKARAN ◽

Melanie R. NILSSON ◽

Anne CLARK

Keyword(s):

Type 2 Diabetes ◽

Amyloid Fibrils ◽

Secretory Granules ◽

Pancreatic Beta Cell ◽

Islet Amyloid Polypeptide ◽

Fibril Formation ◽

Islet Amyloid ◽

Β Sheet

Islet amyloid polypeptide (IAPP), or ‘amylin’, is co-stored with insulin in secretory granules of pancreatic islet β-cells. In Type 2 diabetes, IAPP converts into a β-sheet conformation and oligomerizes to form amyloid fibrils and islet deposits. Granule components, including insulin, inhibit spontaneous IAPP fibril formation in vitro. To determine the mechanism of this inhibition, molecular interactions of insulin with human IAPP (hIAPP), rat IAPP (rIAPP) and other peptides were examined using surface plasmon resonance (BIAcore), CD and transmission electron microscopy (EM). hIAPP and rIAPP complexed with insulin, and this reaction was concentration-dependent. rIAPP and insulin, but not pro-insulin, bound to hIAPP. Insulin with a truncated B-chain, to prevent dimerization, also bound hIAPP. In the presence of insulin, hIAPP did not spontaneously develop β-sheet secondary structure or form fibrils. Insulin interacted with pre-formed IAPP fibrils in a regular repeating pattern, as demonstrated by immunoEM, suggesting that the binding sites for insulin remain exposed in hIAPP fibrils. Since rIAPP and hIAPP form complexes with insulin (and each other), this could explain the lack of amyloid fibrils in transgenic mice expressing hIAPP. It is likely that IAPP fibrillogenesis is inhibited in secretory granules (where the hIAPP concentration is in the millimolar range) by heteromolecular complex formation with insulin. Alterations in the proportions of insulin and IAPP in granules could disrupt the stability of the peptide. The increase in the proportion of unprocessed pro-insulin produced in Type 2 diabetes could be a major factor in destabilization of hIAPP and induction of fibril formation.

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Modulating functional amyloid formation via alternative splicing of the premelanosomal protein PMEL17

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013012 ◽

2020 ◽

Vol 295 (21) ◽

pp. 7544-7553 ◽

Cited By ~ 1

Author(s):

Dexter N. Dean ◽

Jennifer C. Lee

Keyword(s):

Alternative Splicing ◽

Amyloid Fibrils ◽

Limited Proteolysis ◽

Amyloid Formation ◽

Functional Amyloid ◽

Melanin Biosynthesis ◽

Functional Amyloids ◽

Fibril Morphology ◽

Amyloid Assembly ◽

Β Sheet

The premelanosomal protein (PMEL17) forms functional amyloid fibrils involved in melanin biosynthesis. Multiple PMEL17 isoforms are produced, two of which arise from excision of a cryptic intron within the amyloid-forming repeat (RPT) domain, leading to long (lRPT) and short (sRPT) isoforms with 10 and 7 imperfect repeats, respectively. Both lRPT and sRPT isoforms undergo similar pH-dependent mechanisms of amyloid formation and fibril dissolution. Here, using human PMEL17, we tested the hypothesis that the minor, but more aggregation-prone, sRPT facilitates amyloid formation of lRPT. We observed that cross-seeding by sRPT fibrils accelerates the rate of lRPT aggregation, resulting in propagation of an sRPT-like twisted fibril morphology, unlike the rodlike structure that lRPT normally adopts. This templating was specific, as the reversed reaction inhibited sRPT fibril formation. Despite displaying ultrastructural differences, self- and cross-seeded lRPT fibrils had a similar β-sheet structured core, revealed by Raman spectroscopy, limited-proteolysis, and fibril disaggregation experiments, suggesting the fibril twist is modulated by N-terminal residues outside the amyloid core. Interestingly, bioinformatics analysis of PMEL17 homologs from other mammals uncovered that long and short RPT isoforms are conserved among members of this phylogenetic group. Collectively, our results indicate that the short isoform of RPT serves as a “nucleator” of PMEL17 functional amyloid formation, mirroring how bacterial functional amyloids assemble during biofilm formation. Whereas bacteria regulate amyloid assembly by using individual genes within the same operon, we propose that the modulation of functional amyloid formation in higher organisms can be accomplished through alternative splicing.

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Characterization by Nano-Infrared Spectroscopy of Individual Aggregated Species of Amyloid Proteins

Molecules ◽

10.3390/molecules25122899 ◽

2020 ◽

Vol 25 (12) ◽

pp. 2899 ◽

Cited By ~ 3

Author(s):

Jehan Waeytens ◽

Vincent Van Hemelryck ◽

Ariane Deniset-Besseau ◽

Jean-Marie Ruysschaert ◽

Alexandre Dazzi ◽

...

Keyword(s):

Infrared Spectroscopy ◽

Spatial Resolution ◽

Amyloid Fibrils ◽

Thin Layers ◽

Fibrillar Structure ◽

Native Structure ◽

Force Microscopy ◽

Atomic Force ◽

Innovative Tool ◽

Β Sheet

Amyloid fibrils are composed of aggregated peptides or proteins in a fibrillar structure with a higher β-sheet content than in their native structure. To characterize them, we used an innovative tool that coupled infrared spectroscopy with atomic force microscopy (AFM-IR). With this method, we show that we can detect different individual aggregated species from oligomers to fibrils and study their morphologies by AFM and their secondary structures based on their IR spectra. AFM-IR overcomes the weak spatial resolution of usual infrared spectroscopy and achieves a resolution of ten nanometers, the size of isolated fibrils. We characterized oligomers, amyloid fibrils of Aβ42 and fibrils of α-synuclein. To our surprise, we figured out that the nature of some surfaces (ZnSe) used to study the samples induces destructuring of amyloid samples, leading to amorphous aggregates. We strongly suggest taking this into consideration in future experiments with amyloid fibrils. More importantly, we demonstrate the advantages of AFM-IR, with a high spatial resolution (≤ 10 nm) allowing spectrum recording on individual aggregated supramolecular entities selected thanks to the AFM images or on thin layers of proteins.

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Parkinson’s disease is a type of amyloidosis featuring accumulation of amyloid fibrils of α-synuclein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906124116 ◽

2019 ◽

Vol 116 (36) ◽

pp. 17963-17969 ◽

Cited By ~ 24

Author(s):

Katsuya Araki ◽

Naoto Yagi ◽

Koki Aoyama ◽

Chi-Jing Choong ◽

Hideki Hayakawa ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

X Ray Diffraction ◽

Thin Sections ◽

X Ray ◽

Β Sheet ◽

The Brain

Many neurodegenerative diseases are characterized by the accumulation of abnormal protein aggregates in the brain. In Parkinson’s disease (PD), α-synuclein (α-syn) forms such aggregates called Lewy bodies (LBs). Recently, it has been reported that aggregates of α-syn with a cross-β structure are capable of propagating within the brain in a prionlike manner. However, the presence of cross-β sheet-rich aggregates in LBs has not been experimentally demonstrated so far. Here, we examined LBs in thin sections of autopsy brains of patients with PD using microbeam X-ray diffraction (XRD) and found that some of them gave a diffraction pattern typical of a cross-β structure. This result confirms that LBs in the brain of PD patients contain amyloid fibrils with a cross-β structure and supports the validity of in vitro propagation experiments using artificially formed amyloid fibrils of α-syn. Notably, our finding supports the concept that PD is a type of amyloidosis, a disease featuring the accumulation of amyloid fibrils of α-syn.

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Copper stabilizes antiparallel β-sheet fibrils of the amyloid β40 (Aβ40)-Iowa variant

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011955 ◽

2020 ◽

Vol 295 (27) ◽

pp. 8914-8927

Author(s):

Elliot J. Crooks ◽

Brandon A. Irizarry ◽

Martine Ziliox ◽

Toru Kawakami ◽

Tiffany Victor ◽

...

Keyword(s):

Blood Vessels ◽

Amyloid Fibrils ◽

Brain Parenchyma ◽

Amyloid Peptide ◽

Amyloid Angiopathy ◽

Seeded Growth ◽

Amyloid Deposits ◽

Aβ42 Peptide ◽

Β Sheet ◽

The Brain

Cerebral amyloid angiopathy (CAA) is a vascular disorder that primarily involves deposition of the 40-residue–long β-amyloid peptide (Aβ40) in and along small blood vessels of the brain. CAA is often associated with Alzheimer's disease (AD), which is characterized by amyloid plaques in the brain parenchyma enriched in the Aβ42 peptide. Several recent studies have suggested a structural origin that underlies the differences between the vascular amyloid deposits in CAA and the parenchymal plaques in AD. We previously have found that amyloid fibrils in vascular amyloid contain antiparallel β-sheet, whereas previous studies by other researchers have reported parallel β-sheet in fibrils from parenchymal amyloid. Using X-ray fluorescence microscopy, here we found that copper strongly co-localizes with vascular amyloid in human sporadic CAA and familial Iowa-type CAA brains compared with control brain blood vessels lacking amyloid deposits. We show that binding of Cu(II) ions to antiparallel fibrils can block the conversion of these fibrils to the more stable parallel, in-register conformation and enhances their ability to serve as templates for seeded growth. These results provide an explanation for how thermodynamically less stable antiparallel fibrils may form amyloid in or on cerebral vessels by using Cu(II) as a structural cofactor.

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The secondary structural difference between Lewy body and glial cytoplasmic inclusion in autopsy brain with synchrotron FTIR micro-spectroscopy

Scientific Reports ◽

10.1038/s41598-020-76565-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Katsuya Araki ◽

Naoto Yagi ◽

Yuka Ikemoto ◽

Hideki Hayakawa ◽

Harutoshi Fujimura ◽

...

Keyword(s):

Animal Experiments ◽

Lewy Bodies ◽

Structural Difference ◽

Cytoplasmic Inclusion ◽

Cytoplasmic Inclusions ◽

Glial Cytoplasmic Inclusions ◽

Sheet Structure ◽

Structural Differences ◽

Β Sheet ◽

The Brain

Abstract Lewy bodies (LBs) and glial cytoplasmic inclusions (GCIs) are specific aggregates found in Parkinson’s disease (PD) and multiple system atrophy (MSA), respectively. These aggregates mainly consist of α-synuclein (α-syn) and have been reported to propagate in the brain. In animal experiments, the fibrils of α-syn propagate similarly to prions but there is still insufficient evidence to establish this finding in humans. Here, we analysed the protein structure of these aggregates in the autopsy brains of patients by synchrotron Fourier-transform infrared micro-spectroscopy (FTIRM) analysis without extracting or artificially amplifying the aggregates. As a result, we found that the content of the β-sheet structure in LBs in patients with PD was significantly higher than that in GCIs in patients with MSA (52.6 ± 1.9% in PD vs. 38.1 ± 0.9% in MSA, P < 0.001). These structural differences may provide clues to the differences in phenotypes of PD and MSA.

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Analysis of the Secondary Structure of β-Amyloid (Aβ42) Fibrils by Systematic Proline Replacement

Journal of Biological Chemistry ◽

10.1074/jbc.m406262200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52781-52788 ◽

Cited By ~ 118

Author(s):

Akira Morimoto ◽

Kazuhiro Irie ◽

Kazuma Murakami ◽

Yuichi Masuda ◽

Hajime Ohigashi ◽

...

Keyword(s):

Amino Acid ◽

Secondary Structure ◽

Amyloid Fibrils ◽

Amino Acid Residues ◽

Wild Type ◽

Aβ Peptides ◽

Amyloid Peptides ◽

Aggregation Inhibitors ◽

Β Amyloid ◽

Β Sheet

Amyloid fibrils in Alzheimer's disease mainly consist of 40- and 42-mer β-amyloid peptides (Aβ40 and Aβ42) that exhibit aggregative ability and neurotoxicity. Although the aggregates of Aβ peptides are rich in intermolecular β-sheet, the precise secondary structure of Aβ in the aggregates remains unclear. To identify the amino acid residues involved in the β-sheet formation, 34 proline-substituted mutants of Aβ42 were synthesized and their aggregative ability and neurotoxicity on PC12 cells were examined. Prolines are rarely present in β-sheet, whereas they are easily accommodated in β-turn as a Pro-Xcorner. Among the mutants at positions 15-32, only E22P-Aβ42 extensively aggregated with stronger neurotoxicity than wild-type Aβ42, suggesting that the residues at positions 15-21 and 24-32 are involved in the β-sheet and that the turn at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of Aβ42. The C-terminal proline mutants (A42P-, I41P-, and V40P-Aβ42) hardly aggregated with extremely weak cytotoxicity, whereas the C-terminal threonine mutants (A42T- and I41T-Aβ42) aggregated potently with significant cytotoxicity. These results indicate that the hydrophobicity of the C-terminal two residues of Aβ42 is not related to its aggregative ability and neurotoxicity, rather the C-terminal three residues adopt the β-sheet. These results demonstrate well the large difference in aggregative ability and neurotoxicity between Aβ42 and Aβ40. In contrast, the proline mutants at the N-terminal 13 residues showed potent aggregative ability and neurotoxicity similar to those of wild-type Aβ42. The identification of the β-sheet region of Aβ42 is a basis for designing new aggregation inhibitors of Aβ peptides.

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