Inhibitory Activity of Red Panda Islet Amyloid Polypeptide on Human IAPP Aggregation

ABSTRACT

Human islet amyloid polypeptide (hIAPP), a 37‐residue hormone co‐secreted with insulin from pancreatic β‐cells, undergoes self‐assembly into cytotoxic amyloid fibrils in the pathogenesis of type 2 diabetes (T2D). The resulting islet amyloid deposits are implicated in progressive β‐cell dysfunction and loss, making hIAPP aggregation an attractive therapeutic target. Nonhuman islet amyloid polypeptide (IAPP) orthologs have recently emerged as a valuable source of mechanistic insight, with several variants—including raccoon and chicken IAPPs—demonstrating the capacity to inhibit hIAPP fibril formation. Here, the biophysical characterization of red panda ( Ailurus fulgens ) IAPP (rpIAPP) and its effects on hIAPP aggregation are reported. Using thioflavin T fluorescence assays, circular dichroism spectroscopy, and atomic force microscopy, red panda IAPP is found to inhibit hIAPP fibrillation at substoichiometric concentrations, with significant inhibitory activity observed at molar ratios as low as 1:0.5 (hIAPP:rpIAPP). These results position red panda IAPP as one of the more potent nonhuman IAPP inhibitors characterized to date and support its further evaluation as a scaffold for the rational design of peptide‐based therapeutics targeting T2D‐associated amyloidogenesis.

The figure shows the comparison of (a) human islet amyloid polypeptide (hIAPP) self‐assembling into a fibril structure and (b) a model of hIAPP self‐assembling in the presence of red panda islet amyloid polypeptide (IAPP). The figure was produced from PDB file 6Y1A using Swiss PDB viewer software.

Abbreviations

AFM

atomic force microscope

CD

circular dichroism

HFIP

hexafluoroisopropanol

hIAPP

human IAPP

IAPP

islet amyloid polypeptide

rpIAPP

red panda IAPP

ThT

thioflavin T

1. Introduction

An estimated 40.1 million people in the United States (12% of the total population) were estimated to have diabetes in 2023, with up to 95% of those cases being type 2 diabetes (T2D) [1]. In that same year, 115 million American adults were estimated to have prediabetes [1]. While the root cause of diabetes remains to be determined, the pathogenesis of T2D has been linked to the accumulation of toxic amyloid deposits formed by the aggregation of human islet amyloid polypeptide (hIAPP), a 37‐amino‐acid polypeptide secreted from β‐cells in the pancreatic islets of Langerhans [2, 3, 4]. The formation of these amyloid deposits is thought to be correlated with the failure and death of β‐cells during T2D progression [5, 6]. Not only is hIAPP found to be toxic to human cells, it is also found to be toxic to nonhuman organisms. Janson et al. found that when mice, who do not naturally develop T2D, were genetically engineered to express hIAPP, they spontaneously developed diabetes and ultimately experienced pancreatic β‐cell death [7]. While the exact role of hIAPP in β‐cell death remains an active area of inquiry, considerable evidence exists to link hIAPP amyloid to the progression of T2D.

Humans are not the only organisms known to develop diabetes. Other species who express amyloidogenic variants of islet amyloid polypeptide (IAPP) have the propensity to develop diabetes, while organisms with nonamyloidogenic variants of IAPP tend to be resistant to developing the disease [2, 8, 9, 10, 11]. For example, rat IAPP does not aggregate, and rats are not known to spontaneously develop T2D. However, cat IAPP does aggregate into toxic amyloid, and cats are known to develop diabetes. Whether the amyloidogenicity of IAPP is a side effect or a driving force for the progression of T2D, the relationship does appear to exist between aggregation propensity and disease potential.

Known inhibitors of hIAPP aggregation include natural plant extracts such as mint, peppermint, red bell pepper, and thyme [12, 13]. Additionally, flavonoids and poly‐hydroxy phenols have been shown to function as hIAPP aggregation inhibitors [14, 15, 16, 17]. Small peptide and peptidomimetic inhibitors have also emerged as potential amyloid‐inhibitors [18, 19, 20, 21]. Perhaps most interestingly, IAPP variants from nonhuman organisms have been found to inhibit human IAPP aggregation [8, 22]. As Tang et al. describe, there is an emerging class of known small‐peptide hIAPP inhibitors, including both existing IAPP sequences and de novo designs [5]. The raccoon, chicken, and seal variants of IAPP have been found to partially rescue mammalian cells from the cytotoxic effect of human IAPP and inhibit hIAPP aggregation in vitro [8, 22]. IAPPs from raccoon and chicken have been shown to be potent inhibitors of aggregation, retaining inhibitory effects even when mixed with hIAPP in a 1:0.5 M ratio of hIAPP to peptide inhibitor [8].

The molecular mechanism of β‐cell death in T2D and the specific role of hIAPP aggregation remain unknown. However, if hIAPP aggregation leads to the loss of pancreatic tissue and the progression of T2D, inhibiting the earliest stages of hIAPP aggregation could protect pancreatic β‐cells and slow the progression of T2D. Here, red panda IAPP (rpIAPP) and its ability to inhibit hIAPP aggregation are characterized. While rpIAPP alone shows minor ability to self‐aggregate, mixing with hIAPP in a 1:1 M ratio resulted in no detectable amyloid products. This inhibitory action continued with substoichiometric quantities of rpIAPP, with amyloid inhibition as low as 1:0.5 (hIAPP/rpIAPP). These results suggest that rpIAPP may be a suitable template to build nonamyloidogenic peptide inhibitors of hIAPP aggregation.

2. Materials and Methods

2.1. Sample Preparation

Synthetic hIAPP was purchased from Lifetein (Somerset, NJ), and synthetic red panda IAPP was purchased from WatsonBio Sciences (Houston, TX). Peptide purities were above 96%. Stock solutions of each IAPP were prepared by dissolving in hexafluoroisopropanol (HFIP, Sigma Aldrich) to a concentration of 32 μM in a sonicating water bath for 10 min before storing at −80°C.

2.2. Thioflavin T Assay

About 175 μL of the hIAPP alone and red panda IAPP alone in HFIP was thawed to room temperature and pipetted into a borosilicate glass tube. The HFIP was removed to dryness under speed vacuum. Tris buffer (150 μL; 20 mM, pH 7.4) was added to yield a 37‐μM solution.

To prepare samples with 1:1 or 1:0.5 M ratios of hIAPP mixed with red panda IAPP, the above process was repeated with 175 μL of the hIAPP‐HFIP mixture combined with 175 μL or 88 μL, respectively, of red panda IAPP‐HFIP. The HFIP was removed to dryness under speed vacuum. Tris buffer (150 μL; 20 mM, pH 7.4) was then added to yield a 37‐μM solution of hIAPP with the various molar ratios of rpIAPP.

Samples were incubated at 37°C with orbital shaking (200 rpm) to rapidly induce peptide aggregation. About 20 μL of each sample was mixed with 700 μL of thioflavin T (ThT) solution (100 μM in Tris buffer) at time 0, 20, and 30 min from the start of incubation. The thioflavin T fluorescence emission spectrum (Ex_450nm) was recorded using a Hitatchi F‐7000 fluorescence spectrophotometer.

2.3. Atomic Force Microscopy

Samples were prepared as described above. After 30 min of incubation at 37°C with shaking, a 20 μL of aliquot of each IAPP sample was pipetted onto freshly cleaved mica and incubated for 5 min at room temperature. The mica was rinsed with 200 μL of sterile‐filtered dI water and allowed to air‐dry. Samples were imaged using an Asylum Research MFP‐3D Atomic Force Microscope (Asylum Research) set on A/C mode with a 240 μm silicon cantilever (Olympus). Images shown are the raw data with no flattening.

2.4. Circular Dichroism (CD)

Samples for CD analysis were prepared as described above and prepared in Tris buffer (150 μL; 20 mM, pH 7.4) to a final concentration of 74 μM. Final samples included hIAPP alone, red panda IAPP alone, and a 1:1 M ratio mixture of hIAPP to red panda IAPP, each prepared at a final IAPP concentration of 74 μM. After 30 min of incubation at 37°C with shaking at 200 rpm, each sample was transferred to a 1‐mm path length quartz cuvette and scanned from 190 to 250 nm using a Jasco J‐815 CD Spectrophotometer (Jasco Inc.). CD spectra are the average of three repeats recorded at 1‐nm intervals with an averaging time of 3 s and smoothed to improve signal to noise ratio.

3. Results and Discussion

The amino acid sequence of the red panda variant of IAPP (Figure 1) differs from that of human IAPP by five amino acids (indicated in red in Figure 1). Red panda IAPP differs from raccoon IAPP by two residues (positions 17 and 19) [21]. Since red panda IAPP is structurally similar to raccoon IAPP, a potent inhibitor of hIAPP aggregation, this study aimed to quantitate the inhibitory effects of red panda IAPP on hIAPP aggregation.

FIGURE 1.

Comparison of IAPP amino acid sequences from different organisms. Amino acids that differ from human IAPP are indicated in red.

3.1. Amyloid Inhibition Assessed With Thioflavin T Binding

Thioflavin T binding (ThT) assays are a well‐established method of measuring amyloidogenicity [10, 22, 23, 24]. When ThT binds to hIAPP amyloid, the ThT fluoresces brightly at 488 nm. The greater the amount of amyloid aggregation, the more ThT binding and the greater the intensity of fluorescence. ThT binding serves as an indirect measure of amyloidogenicity, enabling comparison between samples of hIAPP, red panda IAPP, and mixtures of the two.

Across all samples, minimal fluorescence was observed at time zero (range: 141.9 ± 27.1 to 165.2 ± 32.7 fluorescence units; Figure 2). No statistically significant differences were detected between groups at this time point, consistent with the conclusion that a period of incubation is required to initiate amyloid formation.

FIGURE 2.

37 μM hIAPP mixed with red panda IAPP with molar ratios indicated. Samples were incubated at 37°C for 30 min with shaking (200 rpm). Fluorescence at 488 nm was recorded at the indicated time points. The average of at least three trials is shown with error bars indicating the standard deviation among trials.

At 20 and 30 min, hIAPP alone exhibited markedly increased fluorescence (Figure 2), indicating significant amyloid formation relative to time zero. At 20 and 30 min, rpIAPP alone showed a minimal increase in fluorescence compared to hIAPP, indicating minimal rpIAPP aggregation under these conditions. This suggests that red panda IAPP is minimally amyloidogenic in isolation, a finding that is consistent with those of Fortin et al. [9].

At both 20 and 30 min, all samples of hIAPP mixed with red panda IAPP were significantly less fluorescent than hIAPP alone (Figure 2), indicating that red panda IAPP, at molar ratios as low as 1:0.5 (hIAPP/red panda IAPP), significantly inhibited hIAPP amyloid formation.

3.2. Identification of Amyloid Fibrils Using Atomic Force Microscopy

Atomic force microscopy produces a topographical image of a sample deposited on freshly cleaved mica, enabling a qualitative assessment of aggregation as well as providing information about the physical structure of any aggregates formed [25, 26, 27]. Used in combination with ThT binding assays, AFM provides both quantitative and qualitative data while enabling detection of false‐positive or false‐negative ThT results.

Representative AFM images are shown in Figure 3. After 30 min of incubation, hIAPP alone displayed large, dense mats of fibers covering the mica surface, consistent with its high amyloidogenicity. After 30 min of incubation, red panda IAPP alone showed formation of small amounts of fibrils, consistent with previous studies [9]. At 1:1 and 1:0.5 M ratios of hIAPP to red panda IAPP, the AFM slides were consistently clear of fibers and amyloid, indicating effective inhibition of aggregation (Figure 3).

FIGURE 3.

AFM images of hIAPP alone, rpIAPP alone, and mixtures of hIAPP and rpIAPP at indicated molar ratios. All scans were collected after 30 min of incubation at 37°C with shaking at 37°C.

3.3. Characterization of β‐Sheet Character Using CD

CD measures the differential absorption of left‐ and right‐circularly polarized light and enables identification of elements of protein secondary structure, such as α‐helices, β‐sheets, and unstructured random coils. CD was used to assess the changes in secondary structure when red panda IAPP was mixed with hIAPP [13, 24, 28]. Figure 4 shows the CD spectra of hIAPP alone, red panda IAPP alone, and a 1:1 M ratio mixture of the two after 30 min of incubation at 37°C with vigorous shaking (extreme conditions for promoting amyloid aggregates of hIAPP). The resulting spectrum for hIAPP alone showed the classic single‐minima around 220 nm for proteins that primarily adopt β‐sheet secondary structures (characteristic of the cross β structure of amyloid fibrils) [29, 30]. For red panda IAPP alone, the spectra showed a single‐minima at 202 nm, the classic spectra associated with unstructured random coil with a minor indication of β‐sheet structure (a second minima at 220 nm). Under these conditions, the majority of red panda IAPP remains unstructured random coil with some β‐sheet character. The clear distinction between the red panda IAPP spectrum and the classic β‐sheet spectrum of hIAPP is consistent with the lower amyloidogenicity of red panda IAPP.

FIGURE 4.

Circular dichroism spectra of hIAPP alone, red panda IAPP alone, and a 1:1 M ratio mixture of hIAPP to red panda IAPP after 30 min of incubation at 37°C and vigorous shaking (200 rpm).

The spectral profile of the 1:1 M mixture of hIAPP with red panda IAPP appears similar to that of red panda IAPP alone. The 1:1 M mixture shows a strong single‐minima at 202 nm but with a more pronounced negative peak near 202 nm (approximately −49.8 mDeg·cm²·dmol⁻¹). This spectrum also shows a flattening near 220 nm with retained negative ellipticity, again suggesting a mixture of β‐sheet and random coil structural elements. The mixture spectrum is markedly distinct from the β‐sheet signature of hIAPP alone, indicating that red panda IAPP inhibits hIAPP aggregation and formation of β‐sheet structure.

A considerable amount of work has focused on the importance of Phe23 (Figure 1) in the self‐aggregation of hIAPP. Phe23 has shown to be a driver of aggregation, appearing to accelerate early‐stage self‐assembly and fibril formation. Previous work has shown that mutations that replace Phe23 with nonaromatic residues (such as Leu or Ala) lead to IAPP variants with drastically decreased amyloid‐forming propensity and cellular toxicity [31, 32, 33]. As such, it is not necessarily a surprise that rpIAPP alone is considerably less amyloidogenic and less toxic than hIAPP alone. What does stand out is the ability of rpIAPP to act as a strong inhibitor of hIAPP aggregation and fibril formation, joining a short list of known peptide inhibitors of hIAPP aggregation [8, 9, 34, 35, 36, 37].

4. Conclusion

While work continues to determine the root cause of T2D, the cytotoxicity of hIAPP and loss of pancreatic β‐cell mass appears to be an integral part of the progression of the disease [38, 39]. Inhibiting the formation of toxic hIAPP amyloid could slow the progression of the disease. With hIAPP excreted extracellularly, it is possible that small peptides, such as hIAPP analogs or animal IAPP variants, could function as therapeutic agents for inhibiting hIAPP amyloidogenicity and protecting cellular viability.

The results presented here provide evidence that red panda IAPP acts as a substoichiometric inhibitor of human IAPP aggregation. At molar ratios as low as 1:0.5 (hIAPP: rpIAPP) and after 30 min of incubation, ThT assays demonstrate a significant reduction in aggregation relative to hIAPP alone. AFM imaging indicates that red panda IAPP prevents hIAPP fibril formation at 1:1 and 1:0.5 M ratios. CD provided direct spectroscopic data on the relative changes in secondary structure of hIAPP mixed with rpIAPP. The CD results showed that hIAPP alone exhibited a clearly defined β‐sheet secondary structure; the spectrum of red panda IAPP and its mixture with hIAPP were consistent with partial random coil character and suggested disruption of the β‐sheet interactions typical of hIAPP aggregation. These results indicate that red panda IAPP functions as a strong inhibitor of hIAPP aggregation under conditions known to promote amyloid formation.

Funding

This work was supported by the National Institutes of Health under grant 1R15DK112172‐02.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.