Article pubs.acs.org/crt
Strong Inhibitory Effect of Heme on hIAPP Fibrillation Jinming Wu,†,∥ Jie Zhao,†,∥ Zhen Yang,†,‡ Hailing Li,*,†,§ and Zhonghong Gao*,†,§ †
School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China ‡ Department of Chemical and Biomolecular Engineering, University of Houston, Houston, Texas 77004, United States § Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, People’s Republic of China ABSTRACT: The deposition of human islet amyloid polypeptide (hIAPP) within β-cells is implicated in the etiology of type 2 diabetes mellitus (T2Dm). It was reported that heme could bind to hIAPP. We speculate that binding may affect the aggregation of hIAPP. In this study, UV−vis spectroscopy was used to detect the interaction pattern between the heme and hIAPP. ThT and Bis-ANS fluorescence assay, circular dichroism spectroscopy, gel electrophoresis assay, and transmission electron microscopy were employed to study the effect of heme on the aggregation of hIAPP. We found that heme dramatically inhibited hIAPP aggregation, even partially dismantled hIAPP aggregates by preventing its conformational changes. Moreover, a similar inhibitory effect was also observed on mutant hIAPP. In the compared group, the inhibitory effects of protoporphyrin on hIAPP and its mutants aggregation were weaker. Similarly, its effect on the dismantlement of the aggregates was also weaker. On the basis of these results, we revealed that the heme iron center was not required for the inhibitory effect on hIAPP but affected the binding affinity of heme to hIAPP. Besides Arg11 and His18, other hydrophobic residues of hIAPP may also play important roles in heme binding. Our results may help to develop an in-depth understanding of the interaction between heme and hIAPP, which would be helpful in designing new therapeutic strategies against T2Dm. found to bind to amyloid β peptide (Aβ), which is an important biomarker of Alzheimer’s disease (AD),19 and the binding dramatically reduces the aggregation of Aβ.20−22 It is worth noting that AD and T2Dm share appreciable common features in their pathologies, such as abnormal iron homeostasis,23 decay of iron regulatory proteins,24 dysfunction of mitochondrial complex IV,25 and so forth. In addition, Mukherjee et al. report that heme binds to hIAPP, and its residues histidine18 (His18) and arginine11 (Arg11) are crucial to heme binding.26 Given these studies, we speculate that the involvement of heme in T2Dm pathology might associate with the influence of heme on hIAPP aggregation. In this study, we first studied the interaction of heme with hIAPP. Then, multiple analytical approaches, including thioflavin-T (ThT) and Bis-ANS binding assay, circular dichroism spectroscopy, transmission electron microscopy, gel electrophoresis assay were employed to investigate the influence of heme on hIAPP aggregation. Our results may provide a molecular link between high plasma heme contents and the prevalence of T2Dm.
1. INTRODUCTION Type 2 diabetes mellitus (T2Dm) is a metabolic disease, and its progression is characterized by peripheral insulin resistance and the deposition of amyloid fibril formation, which is implicated with pancreatic β-cell death, in the extracellular matrix of β-cells in the islets of Langerhans.1,2 Recent research shows that the human islet amyloid polypeptide (hIAPP), also known as human amylin, is a major component of the deposit.3 hIAPP is a 37-residue hormone, synthesized in pancreatic β-cells and cosecreted with insulin. In the normal state, hIAPP acts as a partner to insulin in glucose metabolism.4,5 It has been found that hIAPP can misfold into soluble oligomers and insoluble mature fibrils in the disease state.6−8 The aggregation of hIAPP is regarded as an important contributor to the loss of pancreatic β-cells9 and graft failure after islet transplantion.10,11 Meanwhile, increasing evidence indicates that the soluble oligomers have the potential to induce membrane destabilization and oxidative stress, and thus, it is thought to be the most toxic species to pancreatic β-cells.12−14 Therefore, inhibiting or modulating the formation of hIAPP oligomers may be an attractive strategy for the prevention and treatment of T2Dm. It has been well accepted that body iron stores strongly relate to the risk of T2Dm,15,16 and the prevalence of T2Dm in patients with thalassemia, a disease with high plasma heme contents, is 6−14%.17,18 These findings suggest the possible involvement of heme in T2Dm pathology, but the underlying molecular mechanism remains unclear. Interestingly, heme is © 2017 American Chemical Society
2. EXPERIMENTAL PROCEDURES 2.1. Materials. Hemin (ferriprotoporphyrin IX chloride), PP (protoporphyrin), 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid Received: June 19, 2017 Published: August 8, 2017 1711
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology
test was used for statistical analysis, and p < 0.05 was considered significant.
(Bis-ANS), hexafluoroisopropanol (HFIP), and thioflavin T (ThT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The hIAPP and mutated peptides (Arg11Asn, His18Asn, and double mutants of Arg11Asn and His18Asn) were obtained from Chinese Peptide Company (Hangzhou, China). Peptides were RP-HPLC purified greater than 95%. All solvents and other reagents were of the highest purity commercially available. Deionized water from a Milli-Q system (Millipore, MA, USA) was used for solution preparation. 2.2. Preparation of hIAPP and Heme Stock Solution. The hIAPP and mutated peptides were dissolved in HFIP at a concentration of 1 mg/mL and kept at room temperature for 4 h. The samples were then freeze-dried after sonication for 1 min and stored at −20 °C. Before each experiment, the peptides were redissolved in formic acid and immediately diluted to the required concentration with 100 mM PB (pH 7.4) prior to use. The heme or PP stock solution (10 mM) was made by dissolving in DMSO and then aliquoted and stored in the dark at −20 °C until use. 2.3. UV−Vis Absorption Spectroscopy. For absorption spectrum acquisition, 20 μM hIAPP peptides or mutants were mixed with 10 μM heme in 100 mM PB (pH 7.4). After incubation at room temperature for 5 min, the spectra were recorded on a UV 2550 spectrophotometer (Shimadzu Co., Japan) at room temperature with a 0.5 cm cuvette. 2.4. ThT and Bis-ANS Fluorescence Assay. ThT and Bis-ANS were used to detect the aggregation of hIAPP. Inhibitory effect of heme and PP on hIAPP aggregation was investigated by incubating hIAPP (8 μM) with heme (8 μM) in 100 mM PB (pH 7.4) at 37 °C for 0, 1, 2, 3, 4, 5, and 6 h. The dismantling effect was determined by adding 8 μM heme to the hIAPP aggregates (8 μM), which had already been aggregated for 24 h, and stayed at 37 °C for 30 min. For the ThT fluorescence measurement, 100 μL of sample was added into 300 μL of ThT solution (8 μM) or Bis-ANS solution (8 μM) in 100 mM PB (pH 7.4), and the mixture was incubated at room temperature for 1 min. The fluorescence spectrum was recorded on a RF5301 spectrofluorometer (Shimadzu Co., Japan). ThT fluorescence was measured with excitation wavelength at 440 nm and emission wavelength at 480 nm. Bis-ANS fluorescence was measured with excitation wavelength at 385 nm and emission wavelength at 496 nm. Excitation and emission slit widths were set at 5 and 10 nm, respectively. Additionally, PP was used to replace heme as a control experiment. 2.5. TEM Imaging of hIAPP Aggregates. Briefly, after incubation of hIAPP (15 μM) in the presence of heme (15 μM) for 24 h, 30 μL of each sample was applied to a 200-mesh Formvar-carbon coated copper grid and allowed to absorb for 10 min. The excess solution was then removed, and the grid was washed with water and dried in air. Finally, the grid was stained with 5% uranyl acetate for 5 min and dried in air again. Images were taken using a transmission electron microscope (HITACHI H-7000FA) with an accelerating voltage of 30 kV. 2.6. Gel Electrophoresis Analysis of hIAPP. hIAPP samples (50 μM) were incubated with heme (50 μM) at 37 °C for 24 h. Then, they were mixed with loading buffer and loaded into 4%−12% Nu-PAGE Bis-Tris Protein Gels (Invitrogen) using MES running buffer for electrophoresis. Silver staining was used for visualization.27 2.7. Circular Dichroism Spectroscopy (CD). For CD spectra, a JASCO circular dichroism spectrometer was used, and recording conditions were set at room temperature with a bandwidth of 1 nm, a 1 s response time, and a scan speed of 100 nm/min. For hIAPP aggregate formation, it was prepared at 15 μM in 5 mM PB (pH 7.4) and stayed to aggregate for 0 and 12 h for comparison. Then, 10 μL of heme at 200 μM was coincubated with 30 μL of hIAPP at 200 μM in 460 μL of PB at 5 mM and pH 7.4 under 37 °C for 12 h. The measurements were performed in a cuvette cell with 1 mm path length. The CD spectra were recorded scanning from 190 to 260 nm, and three accumulations were obtained. The relevant baseline was subtracted by running PB alone or PB containing heme as a blank. 2.8. Statistical Analysis. All experiments were carried out at least in triplicate. Results were expressed as the mean ± SEM. Student’s t
3. RESULTS 3.1. Binding of Heme with hIAPP and Its Mutants. The binding of heme with hIAPP could be revealed through the change of its absorption spectrum.26 Accordingly, our results as shown in Figure 1 revealed a sharp increase in the Soret band of
Figure 1. UV−vis absorption spectrum of heme binding with hIAPP and its mutants. Ten micromolar heme was mixed with or without 20 μM hIAPP peptide in 100 mM PB (pH 7.4), and the mixture was incubated at 37 °C for 10 min. Absorption spectra was recorded using 100 mM PB (pH 7.4) as a control.
heme at 412 nm along with a red shift from 385 to 395 nm upon heme binding with hIAPP. When the residues of Arg11 and His18 of hIAPP were individually replaced by Asn, the extent of the Soret band increase of heme absorption spectrum upon binding was observed to be much lower compared to that when binding with wild-type hIAPP. Moreover, the extent of the Soret band increase was further suppressed when both sites were simultaneously replaced by Asn. It was a clear indication that residues of Arg11 and His18 of hIAPP were highly involved in its interaction with heme. It is worth noting that the red shift of heme was consistently observed upon its binding with all three kinds of hIAPP mutants. It also suggested that other residues of hIAPP other than Arg11 and His18 were also involved in heme binding. 3.2. Effect of Heme on the Aggregation and Disaggregation of hIAPP. It is well-known that ThT is a sensitive fluorescent dye that specifically binds with fibrous structures that leads to increased fluorescence.28 Thus, the ThT fluorescence assay was employed for the quantitative analysis of hIAPP fibrils. As shown in Figure 2A, a significant increase of ThT fluorescence emission was observed upon 2 h of incubation with the hIAPP sample, which indicated a pronounced aggregation of hIAPP. Interestingly, heme almost completely inhibited the signal of hIAPP aggregation revealed by nondetectable fluorescence intensity of ThT upon incubation. Meanwhile, residue site mutants of hIAPP and PP, surrogates of heme, were used for comparing experiments. The Arg11Asn and His18Asn mutants both exhibited stronger fluorescence intensity (Figure 2B and C), which indicated a higher degree of aggregation, compared to that of wild-type hIAPP. Moreover, the degree of aggregation was observed to be even higher in the case of mutation of both sites, Arg11Asn and 1712
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology
Figure 2. Aggregation of hIAPP detected by the ThT fluorescence assay. Both the inhibitory effect of heme on hIAPP peptide aggregation (A−D) and its capability of dismantling the hIAPP aggregates (E−H) were presented. The compared groups were conducted via replicating the experiments by replacing the heme with PP.
His18Asn (Figure 2D). Similarly, heme exhibited an inhibitory effect on the aggregation of hIAPP mutants as well. A comparison of experiments using PP as a surrogate of heme showed that the inhibitory effect of PP on hIAPP aggregation was weaker than heme, particularly in the case of hIAPP mutants (Figure 2A−D). Furthermore, the effect of heme on pre-existing aggregates of hIAPP and its mutants was also investigated (Figure 2E−H). It was found that heme dramatically dismantled the hIAPP aggregates as well as the aggregates of its mutants upon coincubation. As for the comparing experiments of PP, the dismantling effect was also observed to be less effective than heme, and the efficiency was even much less in the case of the double-site mutant. These results altogether strongly suggested that heme could effectively inhibit hIAPP aggregation and even dismantle its aggregates
and that both the iron center and porphyrin ring played crucial roles in these effects. Also, residue sites of Arg11 and His18 of hIAPP greatly contributed to its interaction with heme. To confirm the results obtained from the ThT fluorescence assay, the Bis-ANS assay was also used to study the aggregation of hIAPP. Bis-ANS is a hydrophobic fluorescent probe, which specifically binds to solvent-exposed hydrophobic surfaces that lead to an increased fluorescence emission and a blue shift in the emission maximum.29,30 As shown in Figure 3, the results of Bis-ANS showed exactly similar results that heme effectively inhibited hIAPP aggregation and even dismantled its aggregates and that its porphyrin ring and iron center played crucial roles in the interaction with hIAPP. 3.3. TEM Images of hIAPP Fibrils. In this study, we also used transmission electron microscopy to directly visualize the 1713
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology
Figure 3. Aggregation of hIAPP detected by the Bis-ANS assay. Both the inhibitory effect of heme on hIAPP peptide aggregation (A−D) and its capability of dismantling the hIAPP aggregates (E−H) were presented. The compared groups were conducted via replicating the experiments by replacing heme with PP.
aggregates of hIAPP so that the inhibitory and dismantling effect of heme on hIAPP aggregation could be directly evidenced. As shown in the TEM images (Figure 4), hIAPP and its mutants, including the single-site mutants and doublesite mutants, all formed a typical network of amyloid fibrils after 24 h of incubation.31 Upon the coincubation with heme or its surrogate of PP, the fiber formation was obviously suppressed. In the comparisons between heme and PP, the inhibitory effect of heme was much stronger than that of PP. These results were consistent with the ThT and Bis-ANS fluorescence assay results that heme and PP significantly inhibited hIAPP aggregation. The mutants, however, still exhibited numerous formation of large fibrils even in the presence of heme and PP, especially the double-site mutant. Recalling the aforementioned results, replacing the Arg11 and His18 with Asn decreased the binding of heme to hIAPP and suppressed the relevant inhibitory effect
of heme on hIAPP aggregation. The observation in TEM images was consistent with the spectroscopic results. Similar to the spectroscopic experiments, the dismantling effect of heme on hIAPP aggregates was also checked using TEM. As shown in Figure 5, both heme and its surrogate of PP dismantled the hIAPP aggregates. It is worth noting that it was not a complete dismantlement. 3.4. Gel Electrophoresis Assay of hIAPP Oligomerization. As a further confirmation of the heme effect on hIAPP aggregation, the gel electrophoresis assay was utilized to study the oligomerization of hIAPP upon coincubation with heme. In Figure 6A, we found that the mutants exhibit greater extent of aggregation than the wild-type hIAPP, which was in agreement with the results of the ThT and Bis-ANS binding assay and TEM images. We also found that the amounts of molecules at lower molecular weight were greatly increased upon the 1714
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology
Figure 4. Inhibitory effect of heme on hIAPP aggregation revealed by TEM imaging. The amyloid fibrils alone were prepared by incubating 15 μM peptides in 100 mM PBS (pH 7.4) at 37 °C for 24 h. The inhibitory effects of the heme or PP were studied by adding 30 μM heme or PP to 30 μM peptides and coincubated for 24 h. (A) hIAPP; (B) 1:1 mixture of hIAPP and heme; (C) 1:1 mixture of hIAPP and PP; (D,G,J) single mutant Arg11Asn, His18Asn, and double mutant Arg11Asn and His18Asn; (E,H,Q) 1:1 mixture of mutants and heme; and (F,I,L) 1:1 mixture of mutants and PP. Scale bars are 500 nm.
Figure 5. Dismantling effect of heme on hIAPP aggregates revealed by TEM imaging. The dismantlement effects of heme or PP on hIAPP aggregates were studied by adding 30 μM heme or PP to 30 μM hIAPP aggregates and standing for 30 min at room temperature prior to TEM observation. hIAPP aggregates were prepared by incubating 30 μM peptide at 37 °C for 24 h. (Left) hIAPP; (middle) hIAPP and heme; (right) hIAPP and PP. Scale bars are 500 nm.
binding of heme with hIAPP as well as the binding of PP and that the increase was more in the case of heme than PP. It provided additional evidence that heme inhibited the aggregation of hIAPP. Apart from that, the inhibitory effect was relatively suppressed in the case of hIAPP mutants, especially in the case of the double-site mutants. As for the dismantling effect, a consistent result was observed that heme
and PP exhibited a slight dismantling effect on the hIAPP aggregates. 3.5. CD Studies of the Inhibitory Effect of Heme on the Aggregation of hIAPP. To extend our studies on hIAPP aggregation, CD spectroscopy was used. As shown in Figure 7, wild-type hIAPP displayed a random-coil structure with a strong negative peak at around 203 nm. After 12 h of 1715
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology
Figure 6. hIAPP oligomerization studied by the gel electrophoresis assay. (A) The inhibitory effect of heme and PP on hIAPP aggregation. (B) The dismantling effect of heme and PP on hIAPP aggregates.
Figure 8. Proposed link between heme and T2Dm. hIAPP can misfold into oligomers and mature fibrils in disease state. However, when the heme is present, the hIAPP would bind to the heme making it prone to form oligomers. Moreover, the heme also can dismantle the aggregates of hIAPP. Thus, as a result, heme can enhance the amount of hIAPP oligomers and contribute to the develop of T2Dm.
which indicated a corresponding decrease of hIAPP aggregates, upon the binding of heme with hIAPP (Figures 2 and 3). A similar inhibitory effect was also observed in the TEM images (Figure 4), gel electrophoresis (Figure 6), and CD spectrum (Figure 7). To further understand the structure effect of heme, PP was used as a surrogate of heme in comparison experiments. The only difference between the PP and heme is the iron core. In this study, PP was observed to have a similar inhibitory effect on hIAPP fibril formation as heme. So this suggests that the porphyrin ring of heme made a large contribution to its inhibitory effect to the aggregation of hIAPP. However, it is worth noting that the inhibitory effect of PP always tended to be weaker in the absence of the iron core when compared to that of heme. It indicated that the iron core of heme was beneficial to its connection with hIAPP though not required. Furthermore, it was also interesting to note that heme dramatically inhibited the secondary structure change of hIAPP from random coil to β-structure through our CD spectroscopy data (Figure 7). This result indicated that the inhibitory effect of heme on hIAPP aggregation might be operated via disturbing its conformational changes, which was important for fibril formation.33 It is worth noting that the aromatic−aromatic interaction has been proposed to play a crucial role in amyloid fibril formation, particularly for hIAPP.34 hIAPP contains three aromatic residues, Phe15, Phe23, and Tyr37, which make different contributions to the formation of hIAPP fibrils.35−39 Among the three residues, Phe23 is found to be important for the early stage of the development of β-sheet structure.40 Phe23 is close to the heme binding site His18 of hIAPP. So, we speculate that the binding of heme with hIAPP may interact with the residue of Phe23 and then disturb the formation of β-sheet structure that leads to a decrease in hIAPP fibrils formation. We already mentioned that the residues of His18 and Arg11 are essential for the binding of hIAPP to heme. His18 binds with the iron core of heme, and Arg11 forms hydrogen bonds with the propionate side chains of heme.26 These residues may have impact on the binding of hIAPP with heme and do influence the inhibitory effects of heme on amyloid fibril formation of hIAPP. Thus, single-site mutants Arg11Asn and His18Asn as well as double-site mutant with both Arg11 and His18 replaced by Asn were employed to investigate the
Figure 7. Far-UV CD spectra of hIAPP and its aggregates.
incubation, an increase of the negative peak at around 220 nm was observed, which was a strong evidence of β-sheet formation.32 Upon the coincubation with heme and PP, the conformational change of hIAPP by aggregation was restored back to its native state. It suggested that heme and PP greatly inhibited the aggregation of hIAPP from another aspect. Also, a similar phenomenon was observed that heme exhibited a greater effect than PP.
4. DISCUSSION AND CONCLUSION Recent studies reported that high heme iron intake was associated with the high risk of developing T2Dm.17,18 It was also reported that heme could bind to hIAPP and that the residues of Arg11 and His18 played a crucial role in the binding with heme.26 Given the strong relevance of hIAPP aggregation with T2Dm pathology, it becomes interesting to unveil the effect of heme on the aggregation of hIAPP. Our previous results along with others already showed that heme could bind to Aβ and reduce its aggregation, and even dismantle the aggregates.20−22 In addition, accumulated evidence suggested that AD and T2Dm shared many similar etiological and pathological features, and heme was found to be implicated in the development of AD and T2Dm.23−25 Hence, it was quite safe to speculate that heme may have a connection with hIAPP by inhibiting its aggregation. In this study, we used multiple analytical methods to study the effect of heme on hIAPP aggregation. For the first time, we saw through various observations that heme exhibited a great inhibitory effect on the aggregation of hIAPP. For instance, the fluorescence assay exhibited a dramatic decrease of the fluorescence intensity, 1716
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology
as an important contributor to the loss of pancreatic β-cells and graft failure after islet transplantion (Figure 8). These findings unveil the interaction between heme and hIAPP, which would be helpful to understand the association of heme with T2Dm. This study also suggests a possible effective strategy against T2Dm by interfering with heme−hIAPP complex formation or eliminating free heme. Nevertheless, the hypothesis will be further confirmed in our future studies.
involvement of these two residues in the interaction between heme and hIAPP. First, we found that the Arg11 and His18 residues in hIAPP were relevant to the interaction with heme (Figure 1). The mutation at residue sites of Arg11 and His18 would suppress the Soret intensity increase of heme upon binding. Nevertheless, a red shift of the Soret band was still observed even in the case of hIAPP mutants. As for the effect of heme on the hIAPP mutants, a similar inhibitory effect was observed but at a relatively lower extent in the case of hIAPP mutants compared to its native state. Especially, the results in TEM images and gel electrophoresis showed that still appreciable amounts of hIAPP aggregates were observed in the mutant samples upon the addition of heme. It suggested that Arg11 and His18 of hIAPP played an important role in the interaction of heme with hIAPP. We speculated that replacing Arg11 or His18 with Asn may significantly decrease the affinity of hIAPP binding to heme that leads to the reduced capability of heme on inhibiting hIAPP aggregation. Nevertheless, a similar inhibitory effect of heme was still observed in hIAPP mutants. It also indicated that other residues other than Arg11 and His18 were also involved in the interaction of hIAPP with heme, which was in agreement with the UV−vis binding results. As to the comparing experiments of PP, it only partially inhibited the aggregation of single-site mutants and had very little effect on the aggregation of the double-site mutant. In physiological conditions, the amino acids around His18 exhibited negative charges, so we speculated that the iron of heme could interact with these residues. When the His18 was replaced, the iron center of heme was vital to interact with the negatively charged residues that led to good binding affinity of heme toward the mutants. PP suffered a loss of binding affinity in the absence of the iron core and thus exhibited a weaker inhibitory effect on the aggregation of mutants. Apart from the inhibitory effect on aggregation, the dismantling effect on the existing plaques is also quite important. Unfortunately, many inhibitors of hIAPP cannot mediate the dismantling of existing plaques.41,42 Dismantling hIAPP aggregates seems more difficult than inhibiting the aggregation of monomer hIAPP peptides. Therefore, it is necessary to investigate the dismantling effect of heme on hIAPP fibrils in addition to its inhibitory effect on fibril formation. Interestingly, the gel electrophoresis assay clearly showed that the hIAPP fibrils were partially dismantled after the incubation with heme. However, we still observed lots of fibrils in TEM images of hIAPP aggregates after 30 min of incubation with heme. We speculate that heme may only be able to dismantle the small fibers but have little effect on the large fibers. Thus, the dismantling effect of heme on hIAPP aggregates was not as efficient as we expected, as shown in the TEM images (Figure 5). In conclusion, we discovered that heme could remarkably inhibit the aggregation of hIAPP and even partially dismantle the aggregates. The iron center was not required for the inhibitory effect of heme but was beneficial to the binding affinity of heme to hIAPP. Besides, we found that Arg11 and His18 in hIAPP were vital to the interaction with heme. Our studies also indicated that other hydrophobic residues may also play important roles in its interaction with heme. Moreover, our results suggested that the inhibitory effect of heme on hIAPP aggregation may be operated via disturbing the formation of βsheet structure by interacting with other amino acid residues of hIAPP, such as Phe23. By binding to heme, hIAPP is prone to form oligomers, and the formation of oligomers was regarded
■
AUTHOR INFORMATION
Corresponding Authors
*(H.L.) Tel: 86-27-87541025. Fax: 86-27-87543632. E-mail:
[email protected]. *(Z.G.) Tel: 86-27-87543532. Fax: 86-27-87543632. E-mail:
[email protected]. ORCID
Zhonghong Gao: 0000-0002-7878-9801 Author Contributions ∥
J.W. and J.Z. contributed equally to this work.
Funding
This work was supported by grants from the National Natural Science Foundation of China (Nos. 31170808, 31570810 and 31770866) and Natural Science Foundation of Hubei Scientific Committee (2016CFA001). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The Analytical and Testing Center of Huazhong University of Science and Technology is thanked for its help in CD and TEM analysis.
■
ABBREVIATIONS hIAPP, human islet amyloid polypeptide; T2Dm, type 2 diabetes mellitus; Aβ, amyloid β peptide; ThT, thioflavin-T; TEM, transmission electron microscopy; AD, Alzheimer’s disease; TMB, 3,3′,5,5′-tetramethylbenzidine; PP, protoporphyrin; PB, phosphate-buffer; HFIP, hexafluoroisopropanol; CD, circular dichroism; Bis-ANS, 4,4′-dianilino-1,1′-binaphthyl5,5′-disulfonic acid; Arg11Asn, Arg11 to Asn variant of human islet amyloid polypeptide; His18Asn, His18 to Asn variant of human islet amyloid polypeptide
■
REFERENCES
(1) Hebda, J. A., and Miranker, A. D. (2009) The interplay of catalysis and toxicity by amyloid intermediates on lipid bilayers: Insights from type II diabetes. Annu. Rev. Biophys. 38, 125−152. (2) Konarkowska, B., Aitken, J. F., Kistler, J., Zhang, S. P., and Cooper, G. J. S. (2006) The aggregation potential of human amylin determines its cytotoxicity towards islet β-cells. FEBS J. 273, 3614− 3624. (3) Westermark, P., Wernstedt, C., Wilander, E., Hayden, D. W., O’Brien, T. D., and Johnson, K. H. (1987) Amyloid fibrils in human insulinoma and islets of Langerhans of the diabetic cat are derived from a neuropeptide-like protein also present in normal islet cells. Proc. Natl. Acad. Sci. U. S. A. 84, 3881−3885. (4) Westermark, P., Andersson, A., and Westermark, G. T. (2011) Islet amyloid polypeptide, islet amyloid, and diabetes mellitus. Physiol. Rev. 91, 795−826. (5) Nicolls, M. R. (2004) The clinical and biological relationship between type II diabetes mellitus and Alzheimer’s disease. Curr. Alzheimer Res. 1, 47−54. 1717
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology (6) DeToma, A. S., Salamekh, S., Ramamoorthy, A., and Lim, M. H. (2012) Misfolded proteins in Alzheimer’s disease and type II diabetes. Chem. Soc. Rev. 41, 608−621. (7) Brender, J. R., Salamekh, S., and Ramamoorthy, A. (2012) Membrane disruption and early events in the aggregation of the diabetes related peptide IAPP from a molecular prospective. Acc. Chem. Res. 45, 454−462. (8) Wei, L., Jiang, P., Xu, W. X., Li, H., Zhang, H., Yan, L. Y., ChanPark, M. B., Liu, X. W., Tang, K., Mu, Y. G., and Pervushin, K. (2011) The molecular basis of distinct aggregation pathways of islet amyloid polypeptide. J. Biol. Chem. 286, 6291−6300. (9) Lu, Y., Derreumaux, P., Guo, Z., Mousseau, N., and Wei, G. H. (2009) Thermodynamics and dynamics of amyloid peptide oligomerization are sequence dependent. Proteins: Struct., Funct., Genet. 75, 954−963. (10) Potter, K. J., Abedini, A., Marek, P., Klimek, A. M., Butterworth, S., Driscoll, M., Baker, R., Nilsson, M. R., Warnock, G. L., Oberholzer, J., Bertera, S., Trucco, M., Korbutt, G. S., Fraser, P. E., Raleigh, D. P., and Verchere, C. B. (2010) Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts. Proc. Natl. Acad. Sci. U. S. A. 107, 4305−4310. (11) Westermark, G. T., Westermark, P., Berne, C., and Korsgren, O. (2008) Widespread amyloid deposition in transplanted human pancreatic islets. N. Engl. J. Med. 359, 977−979. (12) Zraika, S., Hull, R. L., Udayasankar, J., Aston-Mourney, K., Subramanian, S. L., Kisilevsky, R., Szarek, W. A., and Kahn, S. E. (2009) Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis. Diabetologia 52, 626−635. (13) Quist, A., Doudevski, I., Lin, H., Azimova, R., Ng, D., Frangione, B., Kagan, B., Ghiso, J., and Lal, R. (2005) Amyloid ion channels: A common structural link for protein-misfolding disease. Proc. Natl. Acad. Sci. U. S. A. 102, 10427−10432. (14) Ono, K., Condron, M. M., and Teplow, D. B. (2009) Structure− neurotoxicity relationships of amyloid β-protein oligomers. Proc. Natl. Acad. Sci. U. S. A. 106, 14745−14750. (15) Fernández-Real, J. M., López-Bermejo, A., and Ricart, W. (2002) Cross-talk between iron metabolism and diabetes. Diabetes 51, 2348−2354. (16) Swaminathan, S., Fonseca, V. A., Alam, M. G., and Shah, S. V. (2007) The role of iron in diabetes and Its complications. Diabetes Care 30, 1926−1933. (17) Simcox, J. A., and McClain, D. A. (2013) Iron and diabetes risk. Cell Metab. 17, 329−341. (18) Wang, X., Fang, X., and Wang, F. (2015) Pleiotropic actions of iron balance in diabetes mellitus. Rev. Endocr. Metab. Disord. 16, 15− 23. (19) Masters, C. L., and Selkoe, D. J. (2012) Biochemistry of amyloid β-protein and amyloid deposits in Alzheimer Disease. Cold Spring Harbor Perspect. Med. 2, a006262. (20) Atamna, H., and Frey, W. H., II (2004) A role for heme in Alzheimer’s disease: heme binds amyloid β and has altered metabolism. Proc. Natl. Acad. Sci. U. S. A. 101, 11153−11158. (21) Atamna, H. (2009) Amino acids variations in amyloid-β peptides, mitochondrial dysfunction, and new therapies for Alzheimer’s disease. J. Bioenerg. Biomembr. 41, 457−464. (22) Yuan, C., and Gao, Z. H. (2013) Aβ interacts with both the iron center and the porphyrin ring of heme: mechanism of heme’s action on Aβ aggregation and disaggregation. Chem. Res. Toxicol. 26, 262− 269. (23) Grünblatt, E., Bartl, J., and Riederer, P. (2011) The link between iron, metabolic syndrome, and Alzheimer’s disease. J. Neural Transm. 118, 371−379. (24) Lim, Y. A., Rhein, V., Baysang, G., Meier, F., Poljak, A., Raftery, M. J., Guilhaus, M. L., Ittner, L. M., Eckert, A., and Götz, J. (2010) Aβ and human amylin share a common toxicity pathway via mitochondrial dysfunction. Proteomics 10, 1621−1633.
(25) Atamna, H., Liu, J. K., and Ames, B. N. (2001) Heme deficiency selectively interrupts assembly of mitochondrial complex IV in human fibroblasts: relevance to aging. J. Biol. Chem. 276, 48410−48416. (26) Mukherjee, S., and Dey, S. G. (2013) Heme bound amylin: spectroscopic characterization, reactivity, and relevance to Type 2 Diabetes. Inorg. Chem. 52, 5226−5235. (27) Guivernau, B., Bonet, J., Valls-Comamala, V., Bosch-Morato, M., Godoy, J. A., Inestrosa, N. C., Peralvarez-Marin, A., FernandezBusquets, X., Andreu, D., Oliva, B., and Munoz, F. J. (2016) Amyloidβ peptide nitrotyrosination stabilizes oligomers and enhances nmdarmediated toxicity. J. Neurosci. 36, 11693−11703. (28) Ban, T., Hamada, D., Hasegawa, K., Naiki, H., and Goto, Y. (2003) Direct observation of amyloid fibril growth monitored by thioflavin T fluorescence. J. Biol. Chem. 278, 16462−16465. (29) Lindgren, M., Sorgjerd, K., and Hammarstrom, P. (2005) Detection and characterization of aggregates, prefibrillar amyloidogenic oligomers, and protofibrils using fluorescence spectroscopy. Biophys. J. 88, 4200−4212. (30) Rosen, C. G., and Weber, G. (1969) Dimer formation from 1amino-8-naphthalenesulfonate catalyzed by bovine serum albumin. A new fluorescent molecule with exceptional binding properties. Biochemistry 8, 3915−3920. (31) Gong, H., Zhang, X., Cheng, B., Sun, Y., Li, C. Z., Li, T., Zheng, L., and Huang, K. (2013) Bisphenol A accelerates toxic amyloid formation of human islet amyloid polypeptide: a possible link between Bisphenol A exposure and Type 2 Diabetes. PLoS One 8, e54198. (32) Kai, T. H., Zhang, L., Wang, X. Y., Jing, A. H., Zhao, B. Q., Yu, X., Zheng, J., and Zhou, F. M. (2015) Tabersonine inhibits amyloid fibril formation and cytotoxicity of Aβ (1−42). ACS Chem. Neurosci. 6, 879−888. (33) Nanga, R. P. R., Brender, J. R., Vivekanandan, S., and Ramamoorthy, A. (2011) Structure and membrane orientation of IAPP in its natively amidated form at physiological pH in a membrane environment. Biochim. Biophys. Acta, Biomembr. 1808, 2337−2342. (34) Gazit, E. (2002) A possible role for π-stacking in the selfassembly of amyloid fibrils. FASEB. J. 16, 77−83. (35) Marek, P., Abedini, A., Song, B. B., Kanungo, M., Johnson, M. E., Gupta, R., Zaman, W., Wong, S. S., and Raleigh, D. P. (2007) Aromatic interactions are not required for amyloid fibril formation by islet amyloid polypeptide but so influence the rate of fibril formation and fibril morphology. Biochemistry 46, 3255−3261. (36) Tracz, S. M., Abedini, A., Driscoll, M., and Raleigh, D. P. (2004) Role of aromatic interactions in amyloid formation by peptides derived from human amylin. Biochemistry 43, 15901−15908. (37) Wiltzius, J. J. W., Sievers, S. A., Sawaya, M. R., and Eisenberg, D. (2009) Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process. Protein Sci. 18, 1521−1530. (38) Tu, L. H., and Raleigh, D. P. (2013) Role of aromatic interaction in amyloid formation by islet amyloid polypeptide. Biochemistry 52, 333−342. (39) Tu, L. H., Young, L. M., Wong, A. G., Ashcroft, A. E., Radford, S. E., and Raleigh, D. P. (2015) Mutational analysis of the ability of resveratrol to inhibit amyloid formation by islet polypeptide: critical evaluation of the importance of aromatic inhibitor and histidine− inhibitor interactions. Biochemistry 54, 666−676. (40) Shim, S. H., Gupta, R., Ling, Y. L., Strasfeld, D. B., Raleigh, D. P., and Zanni, M. T. (2009) Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution. Proc. Natl. Acad. Sci. U. S. A. 106, 6614− 6619. (41) Garcia-Alloza, M., Subramanian, M., Thyssen, D., Borrelli, L. A., Fauq, A., Das, P., Golde, T. E., Hyman, B. T., and Bacskai, B. J. (2009) Existing plaques and neuritic abnormalities in APP:PS1 mice are not affected by administration of the gamma-secretase inhibitor LY411575. Mol. Neurodegener. 4, 19−27. (42) Yan, P., Bero, A. W., Cirrito, J. R., Xiao, Q. L., Hu, X. Y., Wang, Y., Gonzales, E., Holtzman, D. M., and Lee, J. M. (2009) 1718
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
Article
Chemical Research in Toxicology Characterizing the appearance and growth of amyloid plaques in APP/ PS1 mice. J. Neurosci. 29, 10706−10714.
1719
DOI: 10.1021/acs.chemrestox.7b00170 Chem. Res. Toxicol. 2017, 30, 1711−1719
