Paraskevi L. Tsiolaki

An N-terminal pro-atrial natriuretic peptide (NT-proANP) 'aggregation-prone' segment involved in isolated atrial amyloidosis

Isolated atrial amyloidosis (IAA) is a common localized form of amyloid deposition within the atria of the aging heart. The main constituents of amyloid fibrils are atrial natriuretic peptide (ANP) and the N‐terminal part of its precursor form (NT‐proANP). An ‘aggregation‐prone’ heptapeptide (114KLRALLT120) was located within the NT‐proANP sequence. This peptide self‐assembles into amyloid‐like fibrils in vitro, as electron microscopy, X‐ray fiber diffraction, ATR FT‐IR spectroscopy and Congo red staining studies reveal. Consequently, remedies/drugs designed to inhibit the aggregation tendency of this ‘aggregation‐prone’ segment of NT‐proANP may assist in prevention/treatment of IAA, congestive heart failure (CHF) or atrial fibrillation (AF).

Structural studies and cytotoxicity assays of “aggregation‐prone” IAPP8–16 and its non‐amyloidogenic variants suggest its important role in fibrillogenesis and cytotoxicity of human amylin

Amyloid deposits to the islets of Langerhans are responsible for the gradual loss of pancreatic β‐cells leading to type II diabetes mellitus. Human mature islet amyloid polypeptide (hIAPP), a 37‐residue pancreatic hormone, has been identified as the primary component of amyloid fibrils forming these deposits. Several individual segments along the entire sequence length of hIAPP have been nominated as regions with increased amyloidogenic potential, such as regions 8–20, 20–29, and 30–37. A smaller fragment of the 8–20 region, spanning residues 8–16 of hIAPP has been associated with the formation of early transient α‐helical dimers that promote fibrillogenesis and also as a core part of hIAPP amyloid fibrils. Utilizing our aggregation propensity prediction tools AmylPred and AmylPred2, we have identified the high aggregation propensity of the 8–16 segment of hIAPP. A peptide analog corresponding to this segment was chemically synthesized and its amyloidogenic properties were validated using electron microscopy, X‐ray fiber diffraction, ATR FT‐IR spectroscopy, and polarized microscopy. Additionally, two peptides introducing point mutations L12R and L12P, respectively, to the 8–16 segment, were chemically synthesized. Both mutations disrupt the α‐helical properties of the 8–16 region and lower its amyloidogenic potential, which was confirmed experimentally. Finally, cytotoxicity assays indicate that the 8–16 segment of hIAPP shows enhanced cytotoxicity, which is relieved by the L12R mutation but not by the L12P mutation. Our results indicate that the chameleon properties and the high aggregation propensity of the 8–16 region may significantly contribute to the formation of amyloid fibrils and the overall cytotoxic effect of hIAPP.

The pentapeptide LQVVR plays a pivotal role in human cystatin C fibrillization

Human cystatin C (HCC) is a low molecular weight member of the cystatin family (type2). HCC consists of 120 amino acids. Normally it is an inhibitor of cysteine proteases, but in pathological conditions it forms amyloid fibrils in brain arteries of young adults. An ‘aggregation‐prone’ pentapeptide (47LQVVR51) was located within the HCC sequence using AmylPred, an ‘aggregation‐prone’ peptide prediction algorithm developed in our lab. This peptide was synthesized and self‐assembled into amyloid‐like fibrils in vitro, as electron microscopy, X‐ray fiber diffraction, Attenuated Total Reflectance Fourier‐Transform Spectroscopy and Congo red staining studies reveal. Thus, the 47LQVVR51 peptide seems to have an important role in HCC fibrillization.

The amyloid interactome: Exploring protein aggregation

Protein-protein interactions are the quintessence of physiological activities, but also participate in pathological conditions. Amyloid formation, an abnormal protein-protein interaction process, is a widespread phenomenon in divergent proteins and peptides, resulting in a variety of aggregation disorders. The complexity of the mechanisms underlying amyloid formation/amyloidogenicity is a matter of great scientific interest, since their revelation will provide important insight on principles governing protein misfolding, self-assembly and aggregation. The implication of more than one protein in the progression of different aggregation disorders, together with the cited synergistic occurrence between amyloidogenic proteins, highlights the necessity for a more universal approach, during the study of these proteins. In an attempt to address this pivotal need we constructed and analyzed the human amyloid interactome, a protein-protein interaction network of amyloidogenic proteins and their experimentally verified interactors. This network assembled known interconnections between well-characterized amyloidogenic proteins and proteins related to amyloid fibril formation. The consecutive extended computational analysis revealed significant topological characteristics and unraveled the functional roles of all constituent elements. This study introduces a detailed protein map of amyloidogenicity that will aid immensely towards separate intervention strategies, specifically targeting sub-networks of significant nodes, in an attempt to design possible novel therapeutics for aggregation disorders.

Tracking the amyloidogenic core of IAPP amyloid fibrils: Insights from micro-Raman spectroscopy

Human islet amyloid polypeptide (hIAPP) is the major protein component of extracellular amyloid deposits, located in the islets of Langerhans, a hallmark of type II diabetes. The underlying mechanisms of IAPP aggregation have not yet been clearly defined, although the highly amyloidogenic sequence of the protein has been extensively studied. Several segments have been highlighted as aggregation-prone regions (APRs), with much attention focused on the central 8-17 and 20-29 stretches. In this work, we employ micro-Raman spectroscopy to identify specific regions that are contributing to or are excluded from the amyloidogenic core of IAPP amyloid fibrils. Our results demonstrate that both the N-terminal region containing a conserved disulfide bond between Cys residues at positions 2 and 7, and the C-terminal region containing the only Tyr residue are excluded from the amyloid core. Finally, by performing detailed aggregation assays and molecular dynamics simulations on a number of IAPP variants, we demonstrate that point mutations within the central APRs contribute to the reduction of the overall amyloidogenic potential of the protein but do not completely abolish the formation of IAPP amyloid fibrils.

αCGRP, another amyloidogenic member of the CGRP family

The Calcitonin-gene related peptide (CGRP) family is a group of peptide hormones, which consists of IAPP, calcitonin, adrenomedullin, intermedin, αCGRP and βCGRP. IAPP and calcitonin have been extensively associated with the formation of amyloid fibrils, causing Type 2 Diabetes and Medullary Thyroid Carcinoma, respectively. In contrast, the potential amyloidogenic properties of αCGRP still remain unexplored, although experimental trials have indicated its presence in deposits, associated with the aforementioned disorders. Therefore, in this work, we investigated the amyloidogenic profile of αCGRP, a 37-residue-long peptide hormone, utilizing both biophysical experimental techniques and Molecular Dynamics simulations. These efforts unravel a novel amyloidogenic member of the CGRP family and provide insights into the mechanism underlying the αCGRP polymerization.

Hexapeptide Tandem Repeats Dictate the Formation of Silkmoth Chorion, a Natural Protective Amyloid

Silkmoth chorion is a fibrous structure composed mainly of two major protein classes, families A and B. Both families of silkmoth chorion proteins present a highly conserved, in sequence and in length, central domain, consisting of Gly-rich tandem hexapeptide repetitive segments, flanked by two more variable N-terminal and C-terminal arms. Primary studies identified silkmoth chorion as a functional protective amyloid by unveiling the amyloidogenic properties of the central domain of both protein families. In this work, we attempt to detect the principal source of amyloidogenicity of the central domain by focusing on the role of the tandem hexapeptide sequence repeats. Concurrently, we discuss a possible mechanism for the self-assembly of class A protofilaments, suggesting that the aggregation-prone hexapeptide building blocks may fold into a triangle-shaped β-helical structure.

Unraveling the aggregation propensity of human insulin C-peptide.

Over the last 20 years, proinsulin C‐peptide emerged as an important player in various biological events. Much time and effort has been spent in exploring all functional features of C‐peptide and recording its implications in Diabetes mellitus. Only a few studies, though, have addressed C‐peptide oligomerization and link this procedure with Diabetes. The aim of our work was to examine the aggregation propensity of C‐peptide, utilizing Transmission Electron Microscopy, Congo Red staining, ATR‐FTIR, and X‐ray fiber diffraction at a 10 mg ml−1 concentration. Our experimental work clearly shows that C‐peptide self‐assembles into amyloid‐like fibrils and therefore, the aggregation propensity of C‐peptide is a characteristic novel feature that should be related to physiological and also pathological conditions.

Mining databases for protein aggregation: a review

Abstract Protein aggregation is an active area of research in recent decades, since it is the most common and troubling indication of protein instability. Understanding the mechanisms governing protein aggregation and amyloidogenesis is a key component to the aetiology and pathogenesis of many devastating disorders, including Alzheimer’s disease or type 2 diabetes. Protein aggregation data are currently found “scattered” in an increasing number of repositories, since advances in computational biology greatly influence this field of research. This review exploits the various resources of aggregation data and attempts to distinguish and analyze the biological knowledge they contain, by introducing protein-based, fragment-based and disease-based repositories, related to aggregation. In order to gain a broad overview of the available repositories, a novel comprehensive network maps and visualizes the current association between aggregation databases and other important databases and/or tools and discusses the beneficial role of community annotation. The need for unification of aggregation databases in a common platform is also addressed.