Article pubs.acs.org/biochemistry
Inhibition of Human Amylin Amyloidogenesis by Human AmylinFragment Peptides: Exploring the Effects of Serine Residues and Oligomerization upon Inhibitory Potency Kalkena Sivanesam and Niels H. Andersen* Department of Chemistry, University of Washington, Seattle, Washington 98195, United States S Supporting Information *
ABSTRACT: To date, fragments from within the amyloidogenicpatch region of human amylin (hAM) have been shown to aggregate independently of the full-length peptide. In this study, we show that under certain conditions, both oligomers of NFGAILSS and the monomeric form are capable of inhibiting the aggregation of the full-length hAM sequence. The inhibition, rather than aggregate seeding, observed with the soluble portion of aged NFGAILSS solutions was particularly striking occurring at far substoichiometric levels. Apparently, the oligomer form of this fragment is responsible for inhibiting the transition from random coil to β-sheet or serves as a disaggregator of hAM β-oligomers. Sequential deletion of the serine residues from NFGAILSS results in a decrease of inhibition, indicating that these residues are important to the activity of this fragment. We, like others, observed instances of α-helix-like CD spectra prior to β-sheet formation as part of the amyloidogenesis pathway. The partially aggregated sample and the fragments studied display spectroscopic diagnostics, suggesting that they slow down the conversion of full-length hAM monomers to cytotoxic oligomers. widely accepted that the mature fibrils were the toxic form of these peptides,7−9 there is mounting evidence that soluble oligomeric forms may be more toxic.10−12 It is therefore very important for inhibitor design to target this form of hAM in order to mitigate β-cell toxicity. In recent years, there have been a number of studies proposing that hAM does not go directly from its random coil conformation to a β-sheet conformation during amyloidogenesis. Instead, these studies claim that hAM adopts an onpath α-helical intermediate. Evidence of this intermediate was seen in multiple studies using various spectroscopic techniques such as circular dichroism (CD), two-dimensional infrared spectroscopy (2D-IR), and nuclear magnetic resonance spectroscopy (NMR).13−17 Crystal structures of hAM bound to maltose binding protein showed that hAM can adopt an α-helical dimer state with aromatic stacking between interstrand F15 residues. Mutation of F15 to residues that were predicted by molecular modeling to increase the formation of a helical dimer resulted in the increased amyloidogenesis of hAM. Meanwhile, substitution of F15 to lysine that would result in charge repulsion at this site lowered the levels of amyloidogenesis.18 Further analysis of this position showed a negative correlation between increasing the β-sheet propensity (using tert-leucine or isoleucine) and the rate of aggregation, while a positive correlation was found when
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ype II diabetes (T2D) is a condition that affects nearly 27 million Americans and almost 400 million people worldwide. Over the last 25 years, the World Health Organization notes that the global prevalence of diabetes has almost doubled. T2D is characterized by the inability of the pancreas to make insulin, typically as a result of the death of βcells. The 37 amino acid peptide human pancreatic amylin (hAM) has been implicated as a possible cause of β-cell death through the process of amyloidogenesis.1,2 Amyloidogenesis occurs when peptides misfold to form fibrils with an ordered cross-β strand structure. 3 Over 40 diseases, including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease are said to be a result of polypeptide amyloidogenesis.4 Human pancreatic amylin (hAM) can be divided into three regions, the N-terminal region that is known to favor an αhelical conformation especially in the presence of membranes, the middle region (20−29), that has been characterized as being the most amyloidogenic portion of the peptide, and the C-terminal region. In the monomeric state, hAM adopts a random coil conformation with a disulfide bridge between residues C2 and C7 along with an amidated C-terminus. There is, however, some evidence that hAM samples helical conformations even in the monomeric state.5 This conformation is enhanced in the presence of fluoroalcohols.6 The typical amyloidogenesis pathway involves the conversion of peptide secondary structure from the native state (typically random coil) to β-sheets through self−self-recognition of monomers. The resulting oligomers continue to grow, forming protofibrils and eventually mature fibrils. While it used to be © XXXX American Chemical Society
Received: August 1, 2017 Revised: September 14, 2017 Published: September 18, 2017 A
DOI: 10.1021/acs.biochem.7b00739 Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
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Article
MATERIALS AND METHODS Peptide Synthesis and Characterization. Human amylin was synthesized and characterized as previously described.24 Peptide fragments were synthesized using standard Fmoc solid phase synthesis on a CEM Liberty Blue microwave assisted peptide synthesizer. Wang resin preloaded with the C-terminal residue was used. Peptides were cleaved from resin using a cocktail containing 95% trifluoroacetic acid (TFA), 2.5% water, and 2.5% triisopropylsilane (TIPS). Water and TIPS serve as radical scavengers. Crude peptides were dissolved in water and acetonitrile and purified using reverse-phase HPLC. (Varian ProStar 220 HPLC, Agilent 21.2 × 50 mm C18 column, 10 mL/min, eluent A: water with 0.1% TFA, eluent B: acetonitrile with 0.085% TFA) The central peak by HPLC was collected and lyophilized. Purity was assessed using a Bruker Esquire Ion Trap Mass Spectrometer. All peptides were purified to an excess of 97% purity and stored as a TFA salt. Amyloidogenesis Assay. Human amylin peptide was dissolved in neat HFIP, affording a final concentration of 5 mM by mass, to make a stock solution. Rather than freeze-drying this stock to obtain monomeric hAM for sample preparation,22,35 the stock solution was diluted to make assay samples with a final concentration of 50 μM hAM in 50 mM pH 2.0 phosphate buffer containing 25 mM NaCl and 1 vol % HFIP. Volumetric serial dilution provided greater hAM concentration accuracy than weighing small quantities of lyophilizate. All fragment peptides were dissolved in 50 mM pH 2.0 phosphate buffer to make a stock solution with a final peptide concentration of ca. 2 mM. To make aged NFGAILSS, the stock solution was allowed to incubate at room temperature (without stirring) for 2 weeks. At the end of 2 weeks, the solution was spun down and the supernatant was removed. The supernatant was then added to hAM assay samples to afford final concentrations between 5 μM and 50 μM, calculated based on the initial concentration of NFGAILSS monomer; the actual concentrations of peptide and/or peptide oligomer were presumably significantly smaller due to aggregate precipitation. For all other peptides, fresh stock solutions were prepared 30− 60 min before the assay was to be run. Fragment stock solution was added to hAM assay sample to afford fragment peptide concentrations between 5 μM and 50 μM. All assay samples were incubated with stirring in 1/2 dram vials with a 7 mm stir bar in a 37 °C water bath. All assay samples contained 350 μL of the 50 μM hAM solution at the onset. For CD measurements, every hour up to 6 h, 40 μL of sample was removed and diluted with 160 μL of 50 mM pH 2.0 phosphate buffer containing 1 vol % HFIP. CD measurements were then taken on a Jasco J-720 circular dichroism instrument using parameters as previously described.36 For ThT measurements, the same assay sample composition as described above was used. At each time point, a 40-μL aliquot was removed and diluted with 150 μL of 50 mM pH 2.0 phosphate buffer containing 1 vol % HFIP. 10 μL of 1.6 mM ThT in 50 mM pH 2.0 phosphate buffer was added to afford a final concentration of 80 μM ThT. Each assay was run in triplicate with staggered collection times. The initial measurement was taken at 0 min for run 1, at 10 min for run 2, and at 20 min for run 3. This allowed for a more populated graph. The reproducibility of our assay is evident in the smoothness of the ThT curve. Percent inhibition by ThT was calculated as
using residues (leucine and norleucine) that promoted helicity.19 Further proof of a correlation between helicity and the amyloidogenesis of hAM comes from studies with lipid membranes. Membranes have been shown to promote α-helix formation by hAM as well as speed up the amyloidogenesis of this peptide.20,21 Helicity has also been studied as a possible stable conformation that could prevent amyloidogenesis. The addition of hexafluoroisopropanol (HFIP) at high concentrations, ≥ 10 vol %, promotes the helicity of hAM6 but, more importantly, inhibits the formation of amyloid fibrils.22 The stabilization of the helix has been proposed to be the mechanism by which insulin prevents hAM from aggregating, both in vitro and in the secretory granule of islet cells. Targeting the helical intermediate and stabilizing it could therefore be an important aspect of inhibitors of hAM amyloidogenesis. At intermediate concentrations, 6−9 vol % HFIP, hAM forms β-oligomers that set to a thixotropic gel.23 In the 1−4 vol % range, HFIP shows a concentration-dependent enhancement of amyloidogenesis with the lag time decreasing as the concentration of HFIP increases.8,24−27 Crystal structures of hAM-insulin dimers show prominent aromatic stacking between F15 of hAM and Y16 of insulin resulting in a large buried surface area that, according to the authors, prevents the transition of hAM to β-sheets.18 However, most peptide inhibitors of hAM target the middle region of hAM between residues 20−29. These residues have been implicated as being the amyloidogenic region of hAM because they are, as fragment peptides, capable of forming fibrils with a similar morphology as full-length hAM.28−30 The 10 residues in this region, SNNFGAILSS contain one of the three aromatic residues of hAM. There is evidence that mutation of the F23 residue to a leucine results in the decrease in rate of amyloidogenesis of full-length hAM.19 Mutation of residue I26 to a proline results in the complete abolishment of amyloid formation in the full-length peptide. The mutant is also capable, on coincubation, of increasing the lag time of wild-type hAM aggregation by a factor of 20 making it a potent inhibitor of amyloid formation.31 N-Methylation of just two residues within this region, G24 and I26, is also a suitable way to create a peptide that does not aggregate. This double-N-methylated mutant, when coincubated with wild-type full-length hAM, was able to completely inhibit amyloidogenesis. The mutant was also able to disaggregate preformed amyloid aggregates.32 Tenidis et al. have shown that all 10 residues in this region are not necessary for fibril formation. Their overlapping hexapeptide library of this region showed that the sequence 23 NFGAIL27 is also capable of forming fibrils, making this the shortest sequence within hAM that is able to do so. Their coincubation studies of full-length hAM with the NFGAIL fragment showed no effect on hAM amyloidogenesis.33 However, it has been shown that oligomeric hAM is capable of seeding monomeric hAM and increasing the rate of aggregation.34 However, little is known about the effects of the seeds formed by the fragments of hAM on the amyloidogenesis of full-length hAM. In this study, we set out to study those effects by coincubating seeds and monomers of a fragment of hAM with the full-length peptide. The studies were prompted by unexpected observations for solutions derived from aged samples of NFGAILSS. B
DOI: 10.1021/acs.biochem.7b00739 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry
Figure 1. (A) CD data tracking the change in secondary structure of hAM alone over time. (B) A graph of ThT measurements of amyloid formation representing the three staggered runs taken over the course of 300 min.
blue-shifted in both spectra (to 214 nm at 60 min, and 219 nm at 120 min). The reason for the shift is unclear. However, these results most likely indicate the presence of an α-helical intermediate before or while β-structure is being formed during the amyloidogenesis of hAM. Higham et al. support the formation of an α-helix intermediate that is converted to βsheet over time in the presence of 1 vol % HFIP in phosphate buffer.22 We selected the NFGAILSS fragment for our study instead of the NFGAIL fragment that had previously been employed by Tenidis et al. as the shortest possible sequence that forms amyloid fibrils. This was due to solubility concerns with the shorter fragment: the addition of serine residues was expected to increase the solubility of the peptide. NFGAILSS was allowed to form seeds by leaving a 2 mM solution to stand at room temperature for 2 weeks. The presence of seeds was verified by the development of a precipitate at the end of 2 weeks. Before being used in an assay, the stock solution was spun down and the supernatant was used. The concentration of “aged fragment peptide” was crudely approximated as the initial concentration of monomer. The addition of
