Structural and Biochemical Studies of the Amyloid-Forming Proteins Human Islet Amyloid Polypeptide and Amyloid-Beta
- Author(s): Krotee, Pascal Alfreda Lombardo;
- Advisor(s): Eisenberg, David S;
- et al.
Amyloid fibrils are associated with several diseases, including Type-II diabetes (T2D) and Alzheimer’s disease (AD). The fibrils observed in each disease are composed of a particular protein; in T2D and AD, fibrils are primarily composed of human islet amyloid polypeptide (hIAPP), and amyloid-β (Aβ) and tau, respectively. Although these fibrils are associated with disease, the link of fibril structure to cytotoxicity remains elusive. Here, we use structural and biochemical studies of these proteins to uncover new insights into structural elements that may be important for cytotoxicity.
For hIAPP, we observe that fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments that compose its amyloid spine. Using the cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED), we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects; however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP. We apply some of what we learned from these studies and combine it with previous structural studies to generate two putative models of full-length hIAPP fibrils.
Using MicroED and inhibitors developed using structure-based design, we discover that the spines of hIAPP (19-29 S20G) and Aβ (24-34) are similar in sequence and structure. The compatibility of the atomic structures prompts a molecular model as to how cross-seeding occurs between Aβ and hIAPP both in vitro and in vivo. Consistent with this observation, the inhibitors, designed against the hIAPP spine, reduce cytotoxicity of both full-length hIAPP and Aβ. However, the mechanisms of action of the inhibitors are different for the two proteins: they reduce hIAPP cytotoxicity by reducing fibril formation, while they reduce Aβ cytotoxicity by reducing some other prefibrillar assembly.
Next, using mass spectrometry and molecular dynamics (MD) simulations, we explore the potential for select segments of Aβ to form cylindrins, a β-barrel-shaped model for a toxic amyloid oligomer. Oligomers are small, soluble precursors to fibrils and are hypothesized to be the toxic type of Aβ aggregate. We observe that several segments, predicted to form cylindrins using Rosetta, form assemblies with similar cross-sections to the original cylindrin. Furthermore, one segment, Aβ 24-34, forms a trimer of dimers that is recognized by the oligomer-specific antibody, A11, an architecture reminiscent of the original cylindrin.
Last, we describe the development of novel peptide-based inhibitors of tau fibril formation developed using a MD-based method. This method reveals that the most effective peptide-based inhibitors reduce fibril formation by competitive inhibition. The peptide-based inhibitors developed using this method may serve as potential pharmaceutical therapeutics for AD and the class of diseases known as tauopathies.
Taken together, these studies provide insight into potentially disease-relevant structures formed by proteins implicated in T2D and AD as well as novel strategies for mitigating such structures. Going forward, these studies may inform our development of more relevant therapeutics for these diseases.