Site-Specific Alkylation of the Islet Amyloid Polypeptide Accelerates Self-Assembly and Potentiates Perturbation of Lipid Membranes


The accumulation of insoluble amyloids in the pancreatic islets is a pathological hallmark of type II diabetes and correlates closely with the loss of β-cell mass. The predominant component of these amyloid deposits is the islet amyloid polypeptide (IAPP). The factors contributing to the conversion of IAPP from a monomeric bioactive peptide hormone into insoluble amyloid brils remain partially elusive. In this study, we investigated the eect of the oxidative non-enzymatic post-translational modication induced by the reactive metabolite 4-hydroxynonenal (HNE) on IAPP aggregation and cytotoxicity. Incubation of IAPP with exogenous HNE accelerated its self-assembly into β-sheet brils and led to the formation of a Michael adduct on the His-18 side chain. To model this covalent modication, the imidazole N(πposition of histidine was alkylated using a close analogue of HNE, the octyl chain. IAPP lipidated at His-18 showed a hastened random coil-to-β-sheet conformational conversion into brillar assemblies with a distinct morphology, a low level of binding to thioavin T, and a high surface hydrophobicity. Introducing an octyl chain on His-18 enhanced the ability of the peptide to perturb synthetic lipid vesicles, to permeabilize the plasma membrane, and to induce the death of pancreatic β-cells. Alkylated IAPP triggered the self-assembly of unmodied IAPP by prompting primary nucleation and increased its capacity to perturb the plasma membrane, indicating that only a small proportion of the modied peptide is necessary to shift the balance toward the formation of proteotoxic species. This study underlines the importance of studying IAPP post-translational modications induced by oxidative metabolites in the context of pancreatic amyloids.

 key physiological hallmark of type 2 diabetes (T2D) consists of the accumulation of amyloid brils in the pancreatic islets.1 While the causative link between amyloid formation and T2D pathogenesis remains the subject of active debates, the progressive deposition of insoluble protein aggregates is closely linked with the gradual loss of insulin- producing β-cells.2 The main proteinaceous constituent of pancreatic amyloid deposits is the islet amyloid polypeptide (IAPP), or amylin IAPP is a 37-residue peptide hormone that is co-synthesized, co-packaged, and co-secreted with insulin by pancreatic β-cells and is implicated in the regulation of satiety and glucose homeostasis. This natively disordered polypeptide that transiently populates helical secondary structures is highly prone to self-assembly into amyloid brils.5 The association between pancreatic amyloid deposits and the progression of T2D has historically led to the postulate that amyloid brils are causing degeneration of β-cells.6 However, recent studies have revealed that the cytotoxicity of IAPP is dependent on its quaternary assembly state and that the toxic proteospecies are mainly the transient nonbrillar oligomers,whereas dened brils are poorly toxic. IAPP rarely deposits in the islets of Langerhans of healthy individuals, although its totoxicity. Identifying these factors, or conditions, of the pancreatic microenvironment modulating IAPP self-assembly and associated toxicity is critical for better dening the role(s) of IAPP deposition in T2D etiology.Studies have revealed that T2D patients are aicted with chronic oxidative stress, as exemplied by the increase in the levels of nitrotyrosine and oxidized low-density lipoprotein and by the decrease in the use of antioxidant defense mecha-


Synthesis  of  N(α)-Fmoc-[N(π)-(octyl)]-L-histidine.Modication of IAPP with an alkane analogue of HNE was performed by alkylation on the histidine imidazole N(π) groupnisms.Several conditions are known to increase oxidative stress in the pancreatic islets, including local inammation, hyperglycemia, high levels of free fatty acid, and lip- otoxicity.2124 In turn, the loss of balance of the redox by adapting a previously reported method.38 Briey, N(α)- Fmoc-N(τ)-(trityl)-L-histidine was methylated using a DMF/ MeOH (50:50) solution containing N,N-diisopropylethylamine (DIPEA, 1.1 equiv) and 2-(6-chloro-1H-benzotriazol-homeostasis can lead to an excessive generation of damagingreactive oxygen species (ROS), which promotes non- enzymatic post-translational modications (PTMs) of proteins, such as methionine oxidation and tyrosine nitration, and the formation of oxidized metabolites.25 Interestingly, accumu-1-yl)-1,1,3,3-tetramethylaminium hexauorophosphate (HCTU, 1.1 equiv). The solvent was removed on a rotary evaporator, and the residue was partitioned with ethyl acetate (EtOAc) and water. The organic extract was washed with aqueous 0.1 M HCl, NaHCO , and NaCl successively, dried lation of intracellular ROS and lipid peroxidation products has (MgSO ), and concentrated been observed for immortalized pancreatic β-cells treated with in vacuo. The N(π)-alkylated exogenous IAPP. In Goto-Kakizaki rats, a non-obese model of T2D, high levels of the oxidative stress markers 8- oxo-2′-deoxyguanosine and 4-hydroxynonenal (HNE) were product was obtained using in situ-generated alkyl triate. To a stirred solution of triic anhydride (1.1 equiv) in CH2Cl2 under nitrogen at −75 °C was added dropwise a solution of 1-octanol (1.1 equiv) and DIEA (1.1 equiv) over 10 min. The measured in the islets of Langerhans. HNE is a prevalent and very toxic lipid-derived aldehyde formed by the oxidation of ω-6 polyunsaturated fatty acids.29 This highly reactive aldehyde is known to covalently modify a diversity of biomacromolecules, altering their physicochemical properties and biological functions.

 HNE spontaneously forms stable reaction mixture was stirred at −75 °C for 20 min. A solution of N(α)-Fmoc-N(τ)-(trityl)-L-histidine methyl ester (0.78 mmol, 1 equiv) in CH2Cl2 was added then dropwise, and the mixture was allowed to return to room temperature (RT) and stirred overnight. The mixture was poured into aqueous NaHCO and then extracted with CH Cl . Deprotection was adducts with proteins by reacting with the nucleophilic groups  of cysteine, histidine, and lysine, via the formation of a Schibase or through Michael addition.29 In diabetic patients aicted with Alzheimer’s disease (AD), it has been observed that neuronal IAPP formed adducts with HNE,31 although the performed by treatment with a solution of CH2Cl2, triuoro- acetic acid (TFA), and triisopropylsilane (TIS) [35:3.5:1 (v/v/ v)] for 2 h at RT. The solvent was removed in vacuo, and the mixture was puried by silica gel column chromatography bygradient elution with a CH Cl /MeOH mixture (from 99:1 to eect of this modication on IAPP aggregation has not been investigated. In addition, the oxidative metabolite HNE has been shown to modulate the self-assembly and/or cytotoxicity of amyloidogenic proteins whose aggregation is associated with (neuro)degenerative diseases. For instance, immunohisto- chemical analyses of brain samples from AD patients revealed high levels of HNE in association with amyloid plaques, while biophysical studies demonstrated that the HNE modication of the amyloid-β (Aβ) peptide promotes its aggregation, increases its anity for lipid membranes, and contributes to its toxicity.Similarly, high levels of HNE−protein conjugates have been reported within nigral neurons of patients aicted with Parkinson’s disease.36 Incubation of α-synuclein with HNE led to multiple Michael adducts, promoting the 90:10). The identity of the compound was validated by high- resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR): 1H NMR (600 MHz, CDCl3) δ 8.01 (s, 1H), 7.69 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 7.2 Hz, 2H), 7.33 (t,

J = 7.2 Hz, 2H), 7.24 (t, J = 7.2 Hz, 2H), 6.90 (s, 1H), 5.67 (d,

J = 6.6 Hz, 1H), 4.51 (d, J = 6.6 Hz, 1H), 4.33 (m, 2H), 4.12

(t, J = 6.6 Hz, 1H), 3.84 (d, J = 7.2 Hz, 2H), 3.70 (s, 3H), 3.09

(ddd, J = 42.6, 10.2, 5.4 Hz, 2H), 1.67 (m, 2H), 1.20 (m, 10H), 0.79 (t, J = 6.6 Hz, 3H); 13C NMR (300 MHz, CDCl3) δ 170.86, 155.82, 143.62, 143.52, 141.35, 127.85, 127.13,

124.96, 122.72, 120.08, 67.21, 53.13, 53.01, 47.05, 46.04,

46.02, 31.66, 30.46, 29.03, 28.96, 26.50, 22.57, 14.04.

Demethylation was performed under nucleophilic conditions to give the nal protected amino acid. Lithium iodide (6 formation of compact oligomers with high mesencephalic cells.toxicity equiv) was added to a stirred solution of N(α)-Fmoc-N(τ)- (octyl)-L-histidine methyl ester in dry EtOAc under argon, and

While it is recognized that pancreatic redox stress is a key feature of T2D, that the covalent addition of HNE modulates the self-assembly of aggregation-prone polypeptides, and that IAPP is covalently modied by HNE in the brain of diabetic patients,31 the impact of HNE on IAPP amyloidogenesis has not been addressed so far. Although no study has directly shown that IAPP is modied by HNE in the pancreatic islets, several pieces of biochemical evidence clearly hint toward this possibility. In this context, this study aimed to investigate the eects of HNE on IAPP aggregation and cytotoxicity. Our results revealed that the main site of HNE carbonylation is the His-18 imidazole ring and that this modication accelerates amyloid formation. By synthesizing an N(π)-alkylated derivative of histidine mimicking HNE conjugation, we investigated the mechanistic contributions of this modication to IAPP self-assembly and its ability to disrupt lipid membranes and to cause pancreatic β-cell death.

the reaction mixture was heated at reux for 20 h. The reaction was quenched by the addition of 10% aqueous HCl, and the mixture extracted with EtOAc. The nal product was puried by silica gel ash chromatography by gradient elution with a CH2Cl2/MeOH mixture (95:5 to 80:20), and the identity of the compound was validated by HRMS.

Peptide Synthesis, Purification, and Characterization. Peptides were synthesized on a solid support based on Fmoc chemistry and the HCTU coupling strategy, as previously described.39 Pseudoproline dipeptide derivatives were incorpo- rated to facilitate peptide synthesis.40 Crude peptides were puried by preparative HPLC using a C18 column with a linear gradient of acetonitrile in H2O/TFA [at 0.6% (v/v)]. Collected fractions were analyzed by analytical HPLC using a C18 column and then conrmed by accurate mass measure- ments by analytical LC-MS using an Agilent 1200 series HPLC instrument coupled to an electrospray time-of-ight mass spectrometer (ESI-TOF). Fractions corresponding to the desired peptide with a purity of >95% were pooled and lyophilized. Peptides were cyclized with 100% DMSO overnight and then diluted to be puried by HPLC a second time.

IAPP Monomerization and Sample Preparation. Peptides were dissolved in 100% hexauoro-2-propanol (HFIP) at a concentration of 1 mg/mL. The HFIP solution was then ltered through a 0.22 μm hydrophilic polyvinylidene diuoride (PVDF) lter and sonicated for 30 min before being lyophilized. The resulting peptide powder was solubilized for a second time in HFIP (1 mg/mL) and sonicated for 30 min, and the solution was aliquoted and lyophilized again. Monomerized  peptides  were  stored  dry  at  −20  °C,  but  for no longer than 4 weeks.

Formation of Amyloid Fibrils. Stock solutions were prepared by dissolving the monomerized lyophilized peptide at a concentration of 50 μM, or as otherwise specied, in 20 mM Tris-HCl (pH 7.4). HNE-IAPP was generated by incubating the peptide at 50 μM with 1 mM HNE in 20 mM Tris-HCl (pH 7.4). Self-assembly occurred over a 48 h period, or as otherwise specied, at RT under quiescent conditions, i.e., without agitation. After the indicated incubation times, aliquots were taken, diluted in Tris-HCl buer, and analyzed by uorescence and circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), and transmission electron microscopy (TEM).

Identification of the Site of HNE Modification by LC-

MS/MS. IAPP (HNE-treated and the respective control) was solubilized in 2% TFA in 20% methanol and digested using pepsin [enzyme:protein ratio of 1:40 (w/w)] in a total volume of   400   μL   overnight   (37   °C,   pH   2−2.5).   Digestion   was stopped by adding 500 μL of water, and the digest was cleaned up on a 1 cm3 (30 mg) Oasis hydrophilic−lipophilic balanced (HLB) SPE cartacetonitrile and injected (20 μL) onto an Aeris PEPTIDE XB- C18 100 mm × 2.1 mm column, with solid core 1.7 μm particles (100 Å) using a Nexera Shimadzu UHPLC system with water (A) and acetonitrile (B), both containing 0.1% modication on cysteine, histidine, and lysine residues. The search was performed for +2 to +4 charge states at a MS tolerance of 0.05 Da on precursor ions and 0.1 Da on fragments. Detected HNE-modied peptides were conrmed upon exact mass and charge state and by being absent in the control sample using PeakView software. A list including the retention time and m/z values of the veried peptides was created, and samples were re-injected for higher-quality MS/ MS data. A second IDA analysis was performed for the four most intense precursor ions (excluded after three occurrences) with an MS/MS accumulation time of 150 ms and a total cycle time of 0.9 s.

Fluorescence Spectroscopy. During a specic incubation period, peptide solutions were mixed with 8-anilino-1- naphthalenesulfonic acid (ANS) or thioavin T (ThT)  to yield nal dye concentrations of 50 and 40 μM, respectively. Excitation wavelengths were 355 and 440 nm for ANS and ThT, respectively. Emission spectra from 385 to 585 nm (ANS) and from 450 to 550 nm (ThT) were recorded. The kinetics of amyloid self-assembly monitored by ThT uorescence  were  carried   out  at  25   °C  in   sealed  96-well black-wall, clear-bottom, and non-binding surface microplates, as previously described.42 The excitation wavelength was set at 440 nm, and the emission at 485 nm was recorded every 10 min. Data obtained from triplicate wells were averaged, corrected by subtracting the corresponding control reaction, and plotted as uorescence versus time.

Circular Dichroism Spectroscopy. During a specic incubation period, peptide solutions were incorporated into a 2 mm path length quartz cell. Far-ultraviolet (far-UV) CD spectra were recorded from 190 to 260 nm using a model J-815 CD spectropolarimeter. The wavelength step was set at 0.5 nm with an average time of 10 s/scan at each wavelength step. Each collected spectrum was background subtracted with peptide-free buer. Raw data were converted to mean residue ellipticity (MRE): formic  acid,  at  a  ow  rate  of  300  μL/min  (40  °C).  The gradient started at 5% B, was held for 2.5 min, and was linearly increased to 30% B at 40 min, to 50% B at 26 min, and to 85% B at 26.5 min. MS and MS/MS spectra were recorded on a Sciex high-resolution quadrupole time-of-ight (Q-TOF) TripleTOF 5600 mass spectrometer equipped with a DuoSpray ion source in positive ion mode set at a source voltage of 5 kV, a source temperature of 500 °C, and GS1/GS2 gas ows of 50 psi, with a declustering potential of 80 V. The instrument performed a survey TOF-MS acquisition from m/z 140 to 1250 (accumulation time of 250 ms), followed by MS/ MS on the 15 most intense precursor ions from m/z 250 to 1250 (excluded for 20 s after two occurrences) using information-dependent acquisition (IDA) with dynamic back- ground subtraction. Each MS/MS acquisition had an accumulation time of 50 ms and a collision energy of 30 ± 10 V. The total cycle time was 1.05 s. MS/MS data were searched against a user-created IAPP. FASTA le by ProteinPilot software using Paragon algorithm41 with a detection protein threshold of unused ProtScore > 0.05 (condence > 10%). The protein search algorithm was changed to consider a probability of 0.80 for HNE

The CD spectra were smoothed with the Savitsky−Golay algorithm using a smoothing moving window of 11 data points.Atomic Force Microscopy. Peptide solutions were diluted in 1% acetic acid to yield a nal concentration of 10 μM and immediately applied to freshly cleaved mica. Micas were washed twice with deionized water and air-dried for 24 h. Images were acquired on a Veeco/Bruker multimode AFM instrument using ScanAsyst-air mode with a silicon tip (2−12 nm tip radius, 0.4 N/m force constant) on a nitride lever. Images were taken at 0.5 Hz and 1024 scans/min.

Transmission Electron Microscopy. Peptide solutions were diluted to 10 μM and applied on a glow-discharged copper carbon-coated 300 mesh grid. Samples were negatively stained with 1.5% (w/v) uranyl formate for 1 min and air-dried for 24 h. Images were recorded using a FEI Tecnai G2 Spirit Twin microscope microscope operating at 120 kV and equipped with a Gatan Ultrascan 4000 4k × 4k CCD camera system.

Critical Aggregation Concentration. Pyrene was solu- bilized in ethanol (1 mM) and diluted in 20 mM Tris-HCl (pH 7.4). Monomerized peptides were solubilized in 100% HFIP and sonicated for 5 min before being solubilized into the

Eect of HNE on IAPP amyloid formation. (A−C) IAPP (50 μM) was incubated at 25 °C under quiescent conditions in 20 mM Tris- HCl buer (pH 7.4) in the presence or absence of 1 mM HNE, and after the indicated incubation time, aliquots were taken to measure (A) the ThT uorescence, (B) the ANS uorescence, and (C) the far-UV CD signal. Representative (D) AFM and (E) TEM images of IAPP incubated alone or in the presence of 20 equiv of HNE. Scale bars of 500 nm for panel D and 200 nm for panel E.pyrene solution, keeping the pyrene concentration at 2 μM and the HFIP concentration at 0.3% (v/v). The excitation wavelength was set at 335 nm, and the emission spectra from 350 to 450 nm were recorded. Fluorescence was measured in an ultramicro 10 mm length cell using a PTI QuantaMaster spectrouorometer. The critical aggregation concentration (CAC) was determined by plotting the ratio of uorescence intensity (373 nm/384 nm) as a function of the concentration and intersection of the two linear ts.43

Leakage of Large Unilamellar Vesicles. Large uni- lamellar vesicles (LUVs) were formed using 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glyc- ero-3-phosphoglycerol (DOPG) at dierent molar ratios and supplemented, or not, with 20% cholesterol (Chol). Lipids were solubilized in 100% chloroform, and the solvent was evaporated with a nitrogen gas stream. The lipid lm was then rehydrated in 20 mM Tris-HCl buer (pH 7.4) containing 70 mM calcein for 30 min. The solution was frozen and thawed ve times before being extruded through a 100 nm nucleopore membrane for 15 cycles. Non-encapsulated calcein was removed using a Sephadex G-25 column. The lipid concentration was determined using an inorganic phosphate detection colorimetric assay. LUV solutions were used immediately, and fresh LUVs were used for each experiment. Peptide solutions were prepared by dissolving the lyophilized powder in 100% DMSO and sonicated for 5 min before being diluted in 20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl. The nal peptide concentration was 25 μM with 0.5% DMSO, and LUVs were added to reach a nal lipid concentration of 500 μM. The control used to determine 100% leakage (Fmax) consisted of calcein and LUVs treated with 0.2% Triton X-100. Dye leakage was reported using the equation

% of leakage =F Fbaseline

Fmax − Fbaseline

Cellular Assays. Rat INS-1 cells were seeded in black-wall, clear-bottom, 96-well plates (tissue culture-treated) at a density of 15,000 cells/well in RPMI-1640 medium supplemented with 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin, 10 mM HEPES, 1 mM sodium

 Identication of site-specic HNE conjugation by LC-MS/MS. (A) High-resolution extracted ion chromatograms for unmodied and HNE-modied peptides formed following HNE treatment of IAPP. The table displays the measured m/z values with respective mass accuracies and retention times as well as peptide condence for HNE-modied sequences. (B) Sequences of human IAPP and His-18 HNE modication via Michael addition. (C) Annotated high-resolution MS/MS spectra of the LVHSSNNFGAIL peptide in its native and HNE-modied forms. pyruvate, and 50 μM β-mercaptoethanol. After incubation for 48  h  at  37  °C  in  5%  CO2,  cells  were  treated  by  the  direct addition of 25 μL of peptide solutions solubilized in 20 mM Tris-HCl (pH 7.4) at 3 times the nal concentration. After treatment with the peptides (monomers or preassembled amyloid brils) for 3, 6, or 24 h, the cellular viability was evaluated using the resazurin reduction assay. The cell viability (in percent) was calculated from the ratio of the uorescence of the treated sample to the control (vehicle-treated).44 The release of cellular lactate dehydrogenase (LDH) in the media associated with membrane perturbation was measured using the LDH cytotoxicity kit. Briey, aliquots of cell culture media were taken and LDH activity was measured using a coupled enzymatic assay resulting in the formation of red formazan, which was detected by measuring the absorbance at 490 nm. LDH release (in percent) was calculated from the ratio of the absorbance of the treated sample to that of the control containing the peptide with lysis buer. For Live/Dead assays, cells were seeded in 12-well plates at a density of 164,000 cells/well for 48 h before treatment with the peptides. The cell viability was analyzed by incubating the cells with 4 × 10−6ethidium homodimer-1 and 2 × 10−6 M calcein-AM for 45 min before imaging with a confocal microscope. Data from at least four assays (with dierent lots) were averaged and expressed as means ± the standard error of the mean (SEM).Statistical Analyses. Statistical analyses were performed with GraphPad Prism 8.0 using the Student’s t test, and statistically signicant dierences were established at p < 0.05 (one asterisk), p < 0.01 (two asterisks), p < 0.001 (three asterisks), and p < 0.0001 (four asterisks).


The Lipid-Derived Aldehyde HNE Hastens IAPP Aggregation into β-Sheet-Rich Fibrils. Previous studiesbhave revealed that the reactive lipid peroxidation products, HNE and 4-oxo-2-nonenal (ONE), modulate the self-assembly of amyloidogenic polypeptides, including the Aβ peptide,α-synuclein,37 and the immunoglobulin light chain. We initially investigated how exogenous HNE aects IAPP aggregation into cross-β quaternary structures. IAPP amyloid formation in the presence or absence of HNE was evaluated over incubation time by monitoring the uorescence of ThT, a small dye whose uorescence increases upon binding to cross- β structures.47 Recording the change in ThT uorescence intensity is a method commonly used to monitor the kineticsof amyloid formation.42 The addition of 20 equiv of HNE accelerated the formation of ThT positive IAPP proteospecies  In fact, in the presence of HNE, the ThT signal peaked after incubation for 6 h under quiescent conditions, while in the presence of the EtOH vehicle control, the plateau of ThT uorescence occurred after incubation for 12 h. Similar results were observed by measuring the formation of hydrophobic clusters by ANS. Interestingly, the presence of HNE in the aggregation mixture induced the formation of IAPP aggregates with greater exposure of hydrophobic clusters, as observed with the ANS uorescence intensity measured after incubation for 24 h . The secondary conformational transition associated with amyloid formation was evaluated by CD spectroscopy. As expected, immediately after its solubilization in Tris-HCl buer, monomerized IAPP displayed a random coil conformation, as inferred from the CD spectra characterized with a single minimum at ∼200 nm

After incubation for 6 h in the absence of HNE, a partial conformational shift was observed, i.e., a decrease in the magnitude of the random coil signal and the appearance of a β- sheet signal at 220 nm. In sharp contrast, after incubation for 6 h in the presence of HNE, IAPP has completely shifted toward a β-sheet-rich secondary structure. It is worth mentioning that Eect of histidine alkylation on IAPP amyloid self-assembly. (A−C) Peptides were incubated at 25 °C in 20 mM Tris-HCl (pH 7.4). (A) ThT uorescence, ANS uorescence, and far-UV CD spectra of 25 μM IAPP and H18-Oc IAPP immediately after solubilization (0 h) and after incubation for 24 h. (B) Representative ThT kinetics of amyloid formation at a low peptide concentration (4 μM). (C) Representative TEM images of IAPP and H18-Oc IAPP immediately after solubilization (0 h) and after incubation for 24 h. The scale bar is 200 nm. (D) Critical aggregation concentration (CAC) of IAPP and H18-Oc determined by pyrene uorescence.

the presence of EtOH [3% (v/v)] used to solubilize HNE accelerated IAPP amyloid formation . The accelerating eect of HNE on IAPP amyloid formation was evaluated by imaging the aggregates assembled after 6 and 24 h by AFM and TEM. As shown in panels D and E of , the presence of HNE promoted the formation of long and linear amyloid brils. Microscopy analyses revealed that the terized by a double bond and a carbonyl group, as for HNE, allows the localization of partial positive charge at the β- carbon, rendering it strongly electrophilic.51 Addition of HNE to Cys, His, and Lys side chains occurs mainly via Michael addition, while Schibase formation involving the Lys primary amine group can also take place. Michael adducts represent the vast majority of HNE-mediated protein modications.

 The presence of HNE in the aggregation mixture did not IAPP sequence contains four residues susceptible to profoundly modify the morphology of the quaternary assemblies, as both samples showed a mixture of twisted and at ribbon brils, characteristic of the polydispersity of IAPP brils assembled in vitro.48,49 Overall, these results indicate that the reactive aldehyde HNE accelerates the aggregation of IAPP into prototypical β-sheet brillar structures.

HNE Selectively Modifies Histidine 18. The high reactivity of HNE is associated with its three functional groups, the aldehyde, the C  C bond, and the hydroxyl. Each of these groups can participate alone, or in sequence, in chemical reactions with dierent macromolecules depending on electron delocalization. A conjugated system charac-HNE-mediated Michael addition: Lys-1, Cys-2, Cys-7, and His-18. Cys-2 and Cys-7 are involved in a disulde bond, which decreases signicantly their reactivity. To identify the main sites of covalent modication, IAPP was incubated in the presence of HNE and analyzed by LC-MS/MS after pepsin digestion. Following database searching, two peptides, both containing His-18, were condently identied as HNE- modied (with 99% condence).  the high- resolution extracted ion chromatograms (±0.01 Da) of each of these two peptides (16LVHSSNNFGAIL27 and 15FLVHSSNN- FGAIL27) in their unmodied and HNE-modied forms in the HNE-treated IAPP sample. The control. Alkylation of His-18 prevents the inhibitory eect of acidic pH on IAPP amyloid formation. (A−C) Peptides were incubated at 25 °C in 20 mM acetate buer (pH 4.5) at 25 μM, and at 0 and 24 h, the aggregation mixture was analyzed by (A) ThT and ANS uorescence and far-UV CD spectroscopy, and (B) TEM (scale bar of 200 nm). (C) Representative ThT kinetics of amyloid formation of IAPP and the H18-Oc derivative (25 μM) in 20 mM acetate buer (pH 4.5).

chromatographic peaks only for the unmodied peptides. The site of modication was conrmed to be His-18 from inspection of the high-resolution MS/MS spectra of these peptides in both forms, as annotated . No other HNE modication sites were detected within the IAPP sequence.

Synthesis of IAPP Specifically Alkylated at His-18. To investigate the molecular mechanisms of the eect of HNE on

IAPP self-assembly, we prepared a substituted IAPP derivative by introducing an octyl chain on the imidazole ring of His-18 (H18-Oc IAPP). This strategy was inspired by the work of Qahwash and colleagues, who used the octanoyl group as a model to study the eects of HNE on Aβ aggregation and cytotoxicity.54 To facilitate the synthesis of H18-alkylated IAPP, we rst modied the imidazole ring in solution before introducing the Fmoc-protected alkylated His during elonga- tion on the solid support. We used a regiospecic synthetic approach to prepare the N(π)-substituted histidine  Briey, after methyl esterication of commercially available N(α)-Fmoc-[N(τ)-Trt]-protected histidine (1), alkylated product 3 was obtained using in situ-generated alkyl triate. Removal of side chain Trt protecting group was performed with TFA to give product 4 and demethylation was performed under nucleophilic con- ditions to give the nal Fmoc-protected His (5) in a satisfactory yield of approximatively 70%. Two-dimensional NMR analysis (NOESY) of product 4 conrmed the alkylation at the N(π) position of the imidazole ring of His . An additional correlation was observed between the proton aindicative of a dipolar interaction. Puried N(α)-Fmoc-[N(π)-octyl]-His (5) was introduced into IAPP synthesis on Rink Amide resin, yielding H18-Oc IAPP.

Site-Specific Alkylation of His-18 Accelerates IAPP Amyloid Formation. Having access to an IAPP derivative specically alkylated on the His-18 imidazole ring, we could now probe the mechanistic eects of HNE conjugation on IAPP self-assembly and associated toxicity. The addition of an octyl   chain   to   the   His-18   side   chain   increases   IAPP hydrophobicity, which required presolubilization of the monomerized peptide in HFIP or DMSO. Thus, IAPP and H18-Oc IAPP were rst solubilized in 100% HFIP or DMSO at 5 mM and then diluted into 20 mM Tris-HCl buer (pH 7.4) to reach a nal peptide concentration of 25 μM and 0.5% (v/v) organic solvent.  alkylation dramatically altered the kinetics of IAPP brillogenesis. In fact, immediately after the addition of H18-Oc IAPP to the aqueous buer (time 0 h), ThT and ANS uorescence positive signals were obtained . In comparison to unmodied IAPP, the proteospecies generated after incubation of the H18-Oc IAPP derivative for 24 h were characterized by low ThT binding capacity and high ANS uorescence, suggesting that the alkylation of IAPP induces the formation of aggregates with a low capacity to bind ThT and high surface hydrophobicity. CD analyses revealed that His-18 alkylation connes IAPP to a secondary β-sheet-rich conformation immediately after solubilization. Interestingly, no prototypical lag phase was observed for H18-Oc IAPP in the ThT kinetics of amyloid formation, even at a low peptide concentration of 4 μM ). By TEM imaging, short (proto)brils could be observed immediately after solubiliza- tion of H18-Oc IAPP, whereas no aggregates were visible for the unmodied peptide. TEM images also revealed macroscopic dierences between IAPP and H18-Oc IAPP brils assembled after incubation for 24 h. While IAPP formed long, thick, and polymorphic brils, H18-Oc brils were shorter and thinner.  Eect of site-specic alkylation of His-18 on IAPP cytotoxicity and lipid membrane perturbation. (A) INS-1E cells were treated with dierent peptide concentrations, and the cell viability was measured after treatment for 24 h. (B−E) INS-1E cells were treated with 25 μM IAPP, or H18-Oc IAPP, and the cell viability and plasma membrane perturbation were measured after dierent incubation times. (C) Quantication of cell viability measured by the Live/Dead assays and (D) representative confocal images of INS-1E cells treated with 25 μM IAPP.

The scale bar is 20 μm. (E) Membrane perturbation evaluated by LDH activity in the cell media. In panels A−C and E, data represent the means ± SEM of at least four individual experiments performed in triplicate. (F) Leakage of calcein from 500 μM DOPC/DOPG (90:10) and DOPC/DOPG/Chol (70:10:20) LUVs induced by 25 μM peptide.average height of 6.1 ± 2.3 nm, while H18-Oc brils displayed an average height of 3.2 ± 1.4 nm . Finally, the CAC was evaluated using pyrene uorescence, which probes the formation of micellar oligomeric aggregates known to correlate closely with the amyloidogenic propensity.55 The observed CAC for H18-Oc was ∼10 times lower than that observed for the unmodied IAPP , indicating that alkylation of the His-18 imidazole ring potentiates IAPP aggregation. It is worth mentioning that brils assembled from H18-Oc IAPP diverge from the brils formed from wild-type IAPP in the presence of HNE. In fact, self-assembly of IAPP in the presence of soluble HNE led to a dense network of ThT positive long brils , whereas H18-Oc brils were shorter with a weak ThT signal . This divergence could be related to the fact that HNE modication on the His- 18 imidazole ring occurs concurrently with IAPP self-assembly and that a small proportion of IAPP molecules is modied prior to nucleation and elongation, in agreement with the MS data .

Moreover, the dierences in the phys- icochemical properties between HNE and the octanoyl chain can also lead to structural and morphological variations within the resulting brils. Additionally, HNE can lead to intra- and intermolecular cross-linkage, which is not the case for the alkyl chain. In addition, both bril preparations, i.e., IAPP incubated in the presence of HNE and H18-Oc IAPP, showed seeding capacities similar to that of brils assembled from the unmodied IAPP.

Alkylated IAPP Self-Assembles at Acidic pH. In vitro at neutral pH, IAPP readily self-assembles into amyloid brils, even at concentrations in the low micromolar range. In sharp contrast, in the secretory granules of pancreatic β-cells, IAPP remains in a quasi-soluble state, although its local concentration is estimated to be roughly between 0.8 and 4 mM.6 It has been proposed that the pH value of 5.5 of the β-cell granules contributes to the inhibition of IAPP aggregation56 In fact, protonation of the imidazole ring of His-18 at acidic pH has been shown to prevent IAPP aggregation, through electrostatic repulsion between adjacent monomers.

 Accordingly, we evaluated how site-specic alkylation modu- lates the propensity of IAPP to self-assemble at acidic pH by incubating the H18-Oc derivative in a pH 4.5 acetate buer. As previously reported, incubation of unmodied IAPP at acidic pH inhibited amyloid formation . On the contrary, H18-Oc IAPP formed linear brils characterized by a high surface hydrophobicity and a β-sheet-rich secondary structure. Nonetheless, the ThT uorescence intensity of H18- Oc IAPP brils assembled at pH 4.5 is reduced, compared to that of the ThT signal measured at neutral pH . This dierence in the nal ThT uorescence intensity could be ascribed to alterations in the anity of the dye and/or binding mode, caused by the protonation of the dimethylamino group of ThT.Thus, as previously reported with the introduction of an ethyl acetate group on His-18,60 alkylation of His-18 prevents the inhibitory eect of acidic pH on IAPP aggregation.

This observation suggests that generation of the reactive lipid aldehyde HNE within the secretory granules could trigger IAPP amyloid formation upon its covalent conjugation at His-18.His-18 Alkylation Potentiates the Cytotoxicity ofIAPP and Its Capacity to Perturb Lipid Membranes. As shown above, site-specic alkylation of the imidazole ring of His-18 hastens IAPP amyloid formation and decreases the critical concentration to form micellar aggregates, likely by increasing peptide hydrophobicity. In turn, this could severely H18-Oc IAPP accelerates unmodied IAPP amyloid formation and potentiates its cytotoxicity. (A and B) IAPP and a mixture of IAPP and H18-Oc IAPP (99:1) were incubated at a nal concentration of 25 μM in 20 mM Tris-HCl buer (pH 7.4), and after incubation for 0, 5, and 24 h, aliquots of the aggregation mixtures were taken and analyzed by (A) ThT and ANS uorescence and far-UV CD spectroscopy. (B) TEM images of 25 μM unmodied IAPP incubated alone or in the presence of 1% H18-Oc IAPP after 5 h. The scale bar is 200 nm. (C) Representative kinetics of amyloid formation of IAPP and an IAPP/H18-Oc IAPP mixture (99:1) at a nal concentration of 25 μM monitored by ThT uorescence. (D) Viability of INS-1E cells after peptide treatment. (E) Perturbation of the plasma membrane of INS-1E cells measured by the LDH activity in the media after dierent treatment times with the peptide. (D and E)

The total peptide concentration for IAPP and IAPP with H18-Oc IAPP is 25 μM. Data represent means ± SEM of at least four individual experiments performed in triplicate. impact the capacity of the peptide to perturb the plasma membrane, which is one of the proposed upstream mechanisms associated with IAPP-induced pancreatic cell death,9 among engagement of the RAGE receptor,61 activation of the pro-apoptotic FAS receptor,62 and defects in activity after these treatment periods . By uorescence microscopy, we observed that the decrease in metabolically active cells in green (calcein-AM) and the loss of plasma membrane integrity in red (ethidium homodimer-1) occur after treatment for 3 h with the alkylated IAPP autophagy.63 Accordingly, we compared the cytotoxicity of the H18-Oc IAPP derivative to that of its unmodied counterpart using β-cell line INS-1E. A concentration-depend- ent decrease in cell viability was observed for both peptides upon treatment for 24 h.

Strikingly, alkylated IAPP was signicantly more toxic than unmodied IAPP for all concentrations evaluated. Next, the cell viability after dierent treatment periods with monomerized peptides was evaluated by means of measurement of the metabolic activity and the Live/Dead assay. Interestingly, 25 μM H18-Oc IAPP decreased the viability of INS-1E cells after incubation for 3 and 6 h, while the unmodied IAPP had no eect on metabolic. In contrast, for the unmodied IAPP, these events were observed only after treatment for 24 h. Next, perturbation of the plasma membrane induced by the peptide was evaluated by measuring the leakage of cytoplasmic LDH. The LDH activity measured in the cell media after treatment for 6 h indicated that both peptides avidly disrupt the plasma membrane . Again, H18-Oc IAPP induced a signicantly higher LDH activity at short incubation times, i.e., 2 and 3 h . This observation suggests that alkylation of IAPP potentiates its capacity to perturb the plasma membrane, either by favoring the formation of membrane-active proteospecies or by increasing the capacity of the monomeric/oligomeric peptides to interact with the lipid bilayer. The capacity of the peptides to disrupt lipid membranes was further explored using synthetic anionic LUVs composed of DOPC and DOPG (9:1), a molar ratio of anionic lipids that closely mimics the composition of the pancreatic β-cell plasma membrane, which is estimated to be ∼2−13 mol % of total phospholipids.64 In the presence of unmodied IAPP, the increase in uorescence, which is associated with a decrease in the level of calcein self- quenching caused by its leakage from LUVs, occurred after incubation for 4 h .

In sharp contrast, H18-Oc IAPP induced an immediate increase in calcein uorescence,with a quasi-absence of a lag phase, and greater perturbation after incubation for 14 h. The same trend was observed when the anionic DOPC/DOPG LUVs were supplemented with 20% cholesterol, which was used to better mimic the lipid composition of the eukaryotic plasma membrane and because cholesterol is known to modulate interaction of IAPP with lipid membranes. As it is known that the percentage of anionic lipids and the presence of cholesterol modulate the capacity of IAPP to induce membrane

ThT kinetics, with the T50 shifting from 6.5 ± 0.1 to 3.2 ± 0.3 h with the addition of 1% H18-Oc IAPP . In addition, the nal ThT uorescence intensity after incubation for 16 h was decreased in the presence of 1% alkylated IAPP, suggestive of a change in the quaternary structure of the heterogeneous brils. These biophysical data indicate that alkylation of His-18 on a subpopulation of the IAPP peptide promotes the aggregation of unmodied IAPP through cross- assembly, which could have implications in the context of HNE conjugation of IAPP in T2D. Accordingly, we evaluated how H18-Oc IAPP modulates the cytotoxicity of its unmodied counterpart using INS-1E cells. Interestingly, a25 μM monomerized IAPP/H18-Oc IAPP mixture (99:1) induced a more pronounced decrease in cell viability after incubation for 24 h in comparison to that with 25 μM IAPP  It is worth mentioning that a 0.25 μM H18-Oc IAPP alone, which corresponds to the 1% molar ratio and is below the CAC for this peptide, had no eect on the viability of INS-1E cells. Evaluation of cytoplasmic LDH release upon treatment with the peptide mixtures revealed a majorpermeabilization,67 we also used dierent lipid compositions of LUVs. Strikingly, for all lipid compositions evaluated, ranging from 2% to 30% of anionic lipids, H18-Oc IAPP showed an enhanced capacity to permeabilize LUVs compared to that of its unmodied counterpart. As observed by ANS uorescence, the H18-Oc brils showed a high level of exposure of hydrophobic clusters , and thus, we evaluated the ability of preassembled H18-Oc brils to induce the perturbation of anionic LUVs. In contrast to brils assembled from the unmodied peptide, H18-Oc brils disrupted synthetic anionic LUVs, with or without supple- mental cholesterol . Nonetheless, the capacity of H18-Oc brils to disrupt synthetic lipid vesicles did not correlate with cytotoxicity toward pancreatic β-cells . This divergence could be ascribed to the complexity and heterogeneity of the cell plasma membrane, including the presence of proteoglycans known to exacerbate IAPP-mediated cytotoxicity.14 Taken together, these observations indicate that the site-specic alkylation of IAPP on His-18 signicantly potentiates its ability to disrupt lipid membranes and to induce cell death.

Alkylated IAPP Accelerates the Self-Assembly of Unmodified IAPP and Increases Its Cytotoxicity. We next investigated if the co-incubation of a small molar ratio of H18-Oc IAPP with its unmodied counterpart could modulate its kinetics of self-assembly and toxicity. IAPP was incubated with the monomerized H18-Oc derivative at a 99:1 molar ratio (IAPP:H18-Oc IAPP; nal concentration of 25 μM), and amyloid formation was followed by ThT and ANS uorescence and CD spectroscopy. As observed by ThT and ANSuorescence, the presence of 1% H18-Oc in the aggregation mixture accelerated unmodied IAPP amyloid formation, as observed by the plateau of uorescence occurring after incubation for 5 h . Similarly, the presence of a small amount of His-18-alkylated IAPP accelerated the random coil-to-β-sheet conformational shift of unmodied IAPP . TEM images of the peptide solutions after incubation for 5 h showed the presence of a network of well- dened and long brils for IAPP incubated with 1% H18-Oc IAPP, while a mixture of short brils and oligomers was observed for the unmodied peptide incubated alone . An accelerating eect of the presence of H18-Oc IAPP on the kinetics of IAPP amyloid formation was also noticed by aprovoked by unmodied IAPP . In particular, the IAPP/H18-Oc IAPP mixture (99:1) induced signicant plasma membrane perturbation after incubation for 2 and 3 h, while the eect of the homogeneous unmodied peptide solution was observed after 24 h. Again, 0.25 μM H18-Oc IAPP alone had not eect on the permeability of the INS-1E membrane , indicating that the eect is associated with cross- assembly. Overall, these results suggest that the modication of His-18 by HNE could trigger amyloid nucleation of nearby unmodied IAPP peptides and potentiate perturbation of lipid membranes.


Studies have reported that the oxidative stress in the pancreatic islets of T2D patients correlates with loss of insulin-secreting β-cells and amyloid deposition, suggesting that ROS and reactive metabolites could play an important role in disease etiology.1 Interestingly, a close association between the reactive metabolite HNE, which is the product of the peroxidation of ω-6 polyunsaturated fatty acids, and protein misfolding has been observed in amyloid-related diseases. Nevertheless, although the peptide hormone IAPP has been reported to form adducts with HNE in vivo,31 the mechanistic contributions of HNE to IAPP aggregation has never been investigated. In the study presented here, we initially showed that the incubation of IAPP with exogenous HNE accelerates the formation of cross-β fibrils and leads to formation of HNE−IAPP covalent adducts. Previous studies have revealed that the eect of HNE on protein aggregation is modulated by the extent of conjugation, by the residues that are modied, and/or by the structural and physicochemical properties of the protein. For instance, modication by HNE promoted the formation of stable oligomers of α-synuclein,68 while HNE conjugation of the immunoglobulin light chain at multiple His residues accelerated the conformational conversion associatedwith the formation of brillar aggregates.46 Herein, we observed that the incubation of IAPP with HNE leads to the formation of a Michael adduct on His-18, whereas no Schibase adducts were detected. HNE exhibits a high reactivity toward thiols and amines, reacting with nucleophilic residues in the following order: Cys > His > Lys.69 Preferential reactivity toward Cys is ascribed to the selectivity principle, i.e.,that soft electrophiles react preferentially with soft nucleo- philes, and is dependent on the thiol−thiolate equilibrium.69 IAPP encompasses two cysteine residues forming a disulde bond within the N-terminal domain, making these thiols considerably less nucleophilic. Reaction of HNE with Lys can lead to Schibase formation, although the kinetics of reaction is slow and the Schibase products are reversible, making Michael adducts predominant,70 as observed in the study presented here.

The multireactivity of HNE makes this aldehyde particularly ecient for the formation of intermolecular polypeptide cross- links through Michael addition at C-3 followed by Schibase formation. Thus, cross-linkage could contribute, with the identied adduct, to the eect of HNE on IAPP aggregation. Nonetheless, to investigate the eects of His-18 alkylation, we decreased the complexity of the system by site-specically introducing an octyl chain on the imidazole ring. The N(α)- Fmoc-[N(π)-octyl]-His derivative was prepared and intro- duced into IAPP elongation on a solid support. Biophysical analyses revealed that His-18 N-π-alkylation accelerates amyloid formation and decreases the IAPP critical aggregation concentration. The lag phase was abolished for H18-Oc IAPP, which forms short β-sheet-rich protobrils immediately after its solubilization. IAPP comprises a highly hydrophobic sequence, with only two charged residues at pH 7.4, and this hydrophobicity is known to drive initial self-recognition.10 The hydrophobicity conferred by the octyl chain likely contributes to intermolecular hydrophobic collapse. This hypothesis is in agreement with a previous study showing that the substitution of His-18 with Gln, a residue with similar volume and hydrophobicity, moderately alters the kinetics of amyloid formation, while the H18L substitution strongly hastens IAPP self-assembly.71 In addition, it has been proposed using molecular dynamics that the accelerating eect of the site- specic modication of His-18 by diethyl pyrocarbonate (DEPC) on IAPP self-assembly could be associated with the modulation of the α-helix secondary structure through destabilization of intramolecular hydrogen bonds.60 In fact, the helical intermediate model proposes that IAPP self- assembly is thermodynamically linked to α-helix formation, in a manner similar to coiled coil formation.

The transient helical structure of IAPP spans from Ala-5 to His-18 and extends to Asn-22 in minor conformers. Accordingly, the His-18 side chain projects on the same α-helix face as Asn-14 and Asn-21 (i, i + 4, and i + 7), likely participating in a hydrogen bond network.74 Whether these helical intermediates are on-pathway to amyloid formation,39 His-18 alkylation decreases the stability of the transient α-helix monomers and/ or helical oligomers, ultimately aecting self-recognition. His-18 alkylation also aects imidazole protonation, which isknown to inuence the kinetics of IAPP self-assembly. For instance, at a pH below the imidazolium pKa, IAPP amyloid formation is slowed, and this has been ascribed to His-18 protonation. Herein, we observed that H18-Oc IAPP, in contrast to its unmodied counterpart, assembles promptly into brils at pH 4.5, which was used to mimic the acidic environment of the pancreatic secretory granules. This observation suggests that IAPP aggregation could be triggered in the secretory granules by the formation of the HNE covalent adduct.

His-18 alkylation not only hastened the IAPP conforma- tional transition associated with self-assembly but also led to brils with dierent morphology and physicochemical properties. Whereas H18-Oc IAPP formed linear brils, these assemblies were thinner and displayed a low level of ThT binding compared to that of their unmodied counterparts. Moreover, H18-Oc brils showed high surface-exposed hydrophobicity, which was associated with their capacity to perturb anionic LUVs. The His imidazole ring enables ve types of interaction (cation−π, ππ stacking, hydrogen−π, hydrogen bond, and coordination complex) and two protonation forms, each having their contributions to protein stability, aggregation, and (supra)molecular structure.76 According to the models of IAPP brils inferred from solid-state NMR (hairpin conformation) and, more recently, from cryo-EM (S-shaped conformation), the His-18 side chain projects outward from the protolament, likely participating in packing into mature brils.

 Studies have revealed that His-18 interacts with Thr-36, or Tyr-37, from the opposite protolament.7 Alternatively, molecular dynamics simula- tion based on the solid-state NMR model of Tycko has proposed that His-18 forms an interlayer hydrogen bond with Ser-19 of the adjacent monomer within the same protola- ment, contributing to elongation.81 The key implication of His- 18 in bril formation has been further highlighted by the fact that the sequence of rodent IAPP (rIAPP), which contains Arg at position 18 and does not brillize, could assemble into amyloids upon the R18H substitution.82 These previous studies suggest that His-18 alkylation likely modulates intermolecular interactions implicated in protolament packing and/or elongation, explaining the morphological dierences between unmodied and H18-Oc IAPP brils.

Several cellular mechanisms associated with the toxicity of

IAPP prebrillar proteospecies have been proposed, including oxidative stress, mitochondrial dysfunction, apoptosis, endo- plasmic reticulum stress, and plasma membrane perturba- tion.It is known that the interaction of IAPP with the outer leaet of the plasma membrane triggers several downstream biochemical events leading to cell death. We observed that the H18-Oc IAPP is more toxic than the unmodied peptide and requires a shorter incubation time to induce INS-1E cell death. By measuring the release of cytoplasmic LDH into the media, we showed that plasma membrane permeabilization occurs signicantly earlier for H18-Oc IAPP, which correlates with its high potency to induce leakage of anionic LUVs. These results could be ascribed to the hastened kinetics of self-assembly of H18-Oc IAPP, to the formation of distinct quaternary species, and/or to potent interactions with lipid membranes. Proposed mechanisms of plasma membrane perturbation by IAPP include the formation of transmembrane pores by oligomeric intermediates and membrane disassembly induced by the mechanical pressure of bril growth at the membrane surface.

The hydrophobicity conferred by His-18 alkyla- tion could cause the formation of less compact oligomers with a high level of exposure of hydrophobic surfaces, as observed with the H18-Oc brils. In turn, this could potentiate their interaction with lipid membranes, as previously reported for oligomers assembled from the N-terminal domain of the HypF protein.88 Alternatively, alkylation of IAPP could prompt bril growth at the membrane surface, inducing disassembly of the INS-1E plasma membrane and lipid vesicles. Our results correlate with a previous study showing that HNE adduction on the Aβ peptide increases its cytotoxicity and potency to disrupt membranes.54 In sharp contrast, modication of His-18 by DEPC led to an IAPP derivative with a low capacity to disrupt the POPG lipid bilayer and reduced cytotoxicity, whichis likely associated with the pro-aggregating eect of DEPC conjugation.60 Co-assembly experiments revealed that a 1% molar ratio of H18-Oc IAPP accelerates the self-assembly of unmodied IAPP and potentiates its capacity to perturb lipid membranes. For instance, permeabilization of the plasma membrane was observed after incubation for 2 h in the presence of an IAPP/H18-Oc IAPP mixture (99:1), in contrast to 24 h for a homogeneous unmodied IAPP solution. This observation suggests that alkylation of His-18 favors peptide− lipid interactions and/or insertion within the lipid bilayers, which in turn serve as a nucleation site for pore formation and/or membrane disassembly by bril growth. This suggests that covalent modication of a small proportion of IAPP by HNE triggers the aggregation of unmodied peptides, ultimately aecting the viability of pancreatic cells.

Overall, this study revealed that HNE promotes  4-Hydroxynonenal IAPP amyloid self-assembly and that the covalent alkylation of His- 18 represents a potential mechanism contributing to pancreatic β-cell degeneration. The data presented here indicate that only a small proportion of alkylated peptide is necessary to make unmodied IAPP highly prone to aggregation and to shift the balance toward cytotoxic proteospecies. Nonetheless, although the selected octyl analogue has a chain length similar to that of HNE and mimics its hydrophobicity, it does not recapitulate HNE polarity and/or potential cross-linking, requiring further mechanistic studies. Despite these limitations, our results highlight the importance of studying spontaneous non- enzymatic post-translational modications induced by oxida- tive metabolites in the context of islet amyloids. This study, which constitutes a rst step toward understanding the eect of HNE in IAPP aggregation and tissue deposition, strongly encourages additional investigation to verify if IAPP is covalently modied by HNE in the pancreatic islets of diabetic patients.


the eect of ethanol on IAPP amyloid formation, characterization of N(α)-Fmoc-[N(π)- (octyl)]-L-histidine methyl ester, analysis of peptides, characterization of amyloid brils, TEM images of IAPP and H18-Oc IAPP monomers at pH 4.5, and lipid Phuong Trang Nguyen − Department of Chemistry, Université du Québec àMontréal, Montreal H3C 3P8, Canada; Quebec Network for Research on Protein Function,


Engineering and Applications, PROTEO,

Mélanie Côté-Cyr − Department of Chemistry, Université du Québec àMontréal, Montreal H3C 3P8, Canada; Quebec Network for Research on Protein Function, Engineering and

Applications, PROTEO,

Nadjib Kihal − Department of Chemistry, Université du Québec àMontréal, Montreal H3C 3P8, Canada; Quebec Network for Research on Protein Function, Engineering and

Applications, PROTEO,

Noé Quittot − Department of Chemistry, Université du Québec àMontréal, Montreal H3C 3P8, Canada; Quebec Network for Research on Protein Function, Engineering and

Applications, PROTEO,

Makan Golizeh − Department of Mathematical and Physical Sciences, Concordia University of Edmonton, Edmonton, AB T5B 4E4, Canada

Lekha Sleno − Department of Chemistry, Université du Québec àMontréal, Montreal H3C 3P8, Canada;


This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2018-06209) and the Canadian Research Chair program (CRC, 950-231440) to S.B. M.B., P.T.N., and

M.C.-C. acknowledge fellowships from NSERC.


The authors declare no competing nancial interest.


The authors acknowledge Dr. Guillaume Charron for his technical assistance.