Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity.

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method 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

Understanding co-polymerization in amyloid formation by direct observation of mixed oligomers

Although amyloid assembly in vitro is commonly investigated using single protein sequences, fibril formation in vivo can be more heterogeneous, involving co-assembly of proteins of different length, sequence and/or post-translational modifications. Emerging evidence suggests that co-polymerization can alter the rate and/or mechanism of aggregation and can contribute to pathogenicity. Electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) is uniquely suited to the study of these heterogeneous ensembles. Here, ESI-IMS-MS combined with analysis of fibrillation rates using thioflavin T (ThT) fluorescence, is used to track the course of aggregation of variants of islet-amyloid polypeptide (IAPP) in isolation and in pairwise mixtures. We identify a sub-population of extended monomers as the key precursors of amyloid assembly, and reveal that the fastest aggregating sequence in peptide mixtures determines the lag time of fibrillation, despite being unable to cross-seed polymerization. The results demonstrate that co-polymerization of IAPP sequences radically alters the rate of amyloid assembly by altering the conformational properties of the mixed oligomers that form

Small molecule probes of protein aggregation

Understanding the mechanisms of amyloid formation and toxicity remain major challenges. Whilst substantial progress has been made in the development of methods able to identify the species formed during self-assembly and to describe the kinetic mechanisms of aggregation, the structure(s) of non-native species, including potentially toxic oligomers, remain elusive. Moreover, how fibrils contribute to disease remains unclear. Here we review recent advances in the development of small molecules and other reagents that are helping to define the mechanisms of protein aggregation in molecular detail. Such probes form a powerful platform with which to better define the mechanisms of structural conversion into amyloid fibrils and may provide the much-needed stepping stone for future development of successful therapeutic agents

A new era for understanding amyloid structures and disease

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-β structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention

Structural and Biochemical Studies of the Amyloid-Forming Proteins Human Islet Amyloid Polypeptide and Amyloid-Beta

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

Structural and Biochemical Studies of the Amyloid-Forming Proteins Human Islet Amyloid Polypeptide and Amyloid-Beta

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