Inter-Species Cross-Seeding: Stability and Assembly of Rat - Human Amylin Aggregates

Citation: Berhanu WM, Hansmann UHE ( Inter-Species Cross-Seeding: Stability and Assembly of Rat - Human Amylin Aggregates Workalemahu M. Berhanu 0 Ulrich H. E. Hansmann 0 Ilia V. Baskakov, University of Maryland School of Medicine, United States of America 0 Department of Chemistry and Biochemistry, University of Oklahoma , Norman, Oklahoma , United States of America Diseases such as type 2 diabetes, Alzheimer's and Parkinson's share as common feature the accumulation of mis-folded disease-specific protein aggregates into fibrillar structures, or plaques. These fibrils may either be toxic by themselves, or act as reservoirs for smaller cytotoxic oligomers. This suggests to investigate molecules as potential therapeutics that either reduce fibril formation or increase fibril stability. One example is rat amylin, which can inhibit aggregation of human amylin, a hallmark of type 2 diabetes. In the present paper, we use molecular dynamics to compare the stability of various preformed aggregates, built out of either human amylin, rat amylin, or mixtures of both. We considered two types of fibrillike oligomers: a single-layer in-register conformation, and a double-layer conformation in which the first U-shaped layer consists of rat amylin and the second layer of human amylin. Our results explain the weak amyloid-inhibiting properties of rat amylin and suggest that membrane leakage due to pore formation is responsible for the toxicity of rat amylin observed in a recent experiment. Together, our results put in question the use of rat amylin or the similar FDA approved drug pramlintide as an inhibitor of human amylin aggregation. They also point to mixed human-rat amylin fibril-like oligomers as possible model-systems for studies of amyloid formation that involve cross-species transmission. - Funding: This study was supported by the National Institutes of Health, R01 grant GM62838. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction In human amyloid diseases, protein mis-folding triggers the formation of amyloid oligomers and fibers that can cause cell death leading to either localized or systemic organ failure [1]. One example is human amylin whose main physiological function is suppression of food intake and inhibition of gastric contractions [2]. Human amylin is one of the most amyloidogenic proteins [3]. Its aggregates damage not only b-cells, leading to the reduction of insulin secretion [4], [5], [6] in type 2 diabetes, but also cells in other organs including kidneys [7], heart [8] and the cerebrovascular system [9]. Likely, the main toxic species are not mature fibers but amyloid oligomers [10], [11], with the fibrils potentially acting as reservoirs for the toxic oligomers. This suggests as potential therapeutics molecules that stabilize fibers and therefore shift the equilibrium from smaller, toxic entities towards the fibrillar state [12], [13]. A candidate for such molecules is rat amylin, which due to its high sequence similarity [14] binds strongly to human amylin, but is not amyloidogenic under physiological conditions [15] (and rats therefore do not develop type 2 diabetes [16,17]). Mixing equal molar concentrations of rat with human amylin leads to a deposition of the non-aggregating rat amylin onto human amylin fibrils resulting in a weak aggregation inhibitor activity [18]. However, the interaction mechanisms that stabilizes these mixed amyloid fibrils are not known, as their structures are difficult to characterize. In the present study, we use multiple longtime molecular dynamics simulations [19], [20], [21] to probe the mechanism by which the non-aggregating rat amylin can grow on the surface of human amylin. For this purpose, we investigate the contribution of specific b-strand to b-stand and b-sheet to b-sheet interactions on the elongation and lateral growth of single and double layer models (with both C-terminalC-terminal and NterminalN-terminal interfaces) of human amylin, rat amylin and mixed rat-amylin oligomers. Our aim is to probe what types of intermolecular interactions reduce the cross species barrier and encourage cross-seeding of human and rat amylin fibril-like oligomers. Such molecular insight may not only help with the rational design of components that improve upon rat amylins inhibitory effects on human amylin aggregation, but also lead to a better understanding of the mechanism of cross-seeding in amyloid diseases that are caused by cross-species transmission. Structural Models Details Both human and rat amylin are built out of 37 residues, of which the first 17 residues (the N-terminal region) are identical in both species, including the two positively charged residues, K1 and R11. The most prominent difference in sequence is the presence of three prolines (which are known to break b-strands) in the Cterminus of rat amylin, at positions 25, 28 and 29 [16]. At position 23, phenylalanine, an aromatic residue, is replaced in rat amylin with the aliphatic leucine. The histidine at position 18 in human amylin is replaced in rat amylin by another basic residue, arginine; and the aliphatic isoleucine at position 26 by valine, which is also aliphatic. As of today, no one has crystallized full-length human amylin. Amyloid fibrils exhibit polymorphism due to differences in the packing at the interface between the two proto-filaments. This polymorphism is also reflected by the variety of fibril models of amylin [27]. Early models are made out of three b-strands in a monomer [22], [23], but the most recent high-resolution amylin fibril structures are U-shaped and formed by only two b-strands. Examples are the models proposed by Wiltzius et al. [24], Luca et al. [25], and Bedrood et al. [26]. The X-ray derived models differ only slightly in the details of side-chain packing and have been shown to be more stable than the NMR Tycko model [28], [29], [30], [31]. For instance, previous molecular dynamics simulations indicate that these X-ray models [28], [29], [30] have more closely interlocked side chains of the b-strands that tighten the binding of two b-sheets making them more compact and stable than the solid state NMR model proposed by the Tycko group. The topology of these X-ray models is similar to that reported by Luca et al [25]) which is based on solid state NMR. Note that the U-shaped human amylin structure is similar to recent fibril models determined from brain tissue of patients. We believe that this lends support for the X-ray model as the most likely candidate structure in investigations of the mechanism which stabilizes the fibers [32]. For these reasons, we use it as start structure [24] in our study. The full-length X-ray human amylin fibril model has a characteristic U-shaped b-strand-loop-b-strand motif and is formed from the atomic structure of segm (...truncated)