Nikolaos N. Louros

Structural Analysis of Peptide-Analogues of Human Zona Pellucida ZP1 Protein with Amyloidogenic Properties: Insights into Mammalian Zona Pellucida Formation

Zona pellucida (ZP) is an extracellular matrix surrounding and protecting mammalian and fish oocytes, which is responsible for sperm binding. Mammalian ZP consists of three to four glycoproteins, called ZP1, ZP2, ZP3, ZP4. These proteins polymerize into long interconnected filaments, through a common structural unit, known as the ZP domain, which consists of two domains, ZP-N and ZP-C. ZP is related in function to silkmoth chorion and in an evolutionary fashion to the teleostean fish chorion, also fibrous structures protecting the oocyte and embryo, that both have been proven to be functional amyloids. Two peptides were predicted as ‘aggregation-prone’ by our prediction tool, AMYLPRED, from the sequence of the human ZP1-N domain. Here, we present results from transmission electron microscopy, X-ray diffraction, Congo red staining and attenuated total reflectance Fourier-transform infrared spectroscopy (ATR FT-IR), of two synthetic peptide-analogues of these predicted ‘aggregation-prone’ parts of the human ZP1-N domain, that we consider crucial for ZP protein polymerization, showing that they both self-assemble into amyloid-like fibrils. Based on our experimental data, we propose that human ZP (hZP) might be considered as a novel, putative, natural protective amyloid, in close analogy to silkmoth and teleostean fish chorions. Experiments are in progress to verify this proposal. We also attempt to provide insights into ZP formation, proposing a possible model for hZP1-N domain polymerization.

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.

Structural studies of “aggregation‐prone” peptide‐analogues of teleostean egg chorion ZPB proteins

Egg envelopes of vertebrates are composed of a family of proteins called zona pellucida (ZP) proteins, which are distinguished by the presence of a common structural polymerizing motif, known as ZP domain. Teleostean fish chorion is a fibrous structure, consisting of protein members of the ZPB/ZP1 and the ZPC/ZP3 families, which are incorporated as tandemly repeating heterodimers inside chorion fibers. Computational analysis of multiple ZPB/ZP1 proteins from several teleostean species, reveals two potential “aggregation‐prone” sequence segments, forming a specific polymerization interface (AG interface). These two peptides were synthesized and results are presented in this work from transmission electron microscopy, Congo red staining, X‐ray fiber diffraction and ATR FT‐IR, which clearly display the ability of these peptides to self‐aggregate, forming amyloid‐like fibrils. This, most probably implies that the AG interface of ZPB/ZP1 proteins plays an important role for the formation of the repeating ZPB‐ZPC heterodimers, which constitute teleostean chorion fibrils.

A common 'aggregation-prone' interface possibly participates in the self-assembly of human zona pellucida proteins.

Human zona pellucida (ZP) is composed of four glycoproteins, namely ZP1, ZP2, ZP3 and ZP4. ZP proteins form heterodimers, which are incorporated into filaments through a common bipartite polymerizing component, designated as the ZP domain. The latter is composed of two individually folded subdomains, named ZP‐N and ZP‐C. Here, we have synthesized six ‘aggregation‐prone’ peptides, corresponding to a common interface of human ZP2, ZP3 and ZP4. Experimental results utilizing electron microscopy, X‐ray diffraction, ATR FT‐IR spectroscopy and polarizing microscopy indicate that these peptides self‐assemble forming fibrils with distinct amyloid‐like features. Finally, by performing detailed modeling and docking, we attempt to shed some light in the self‐assembly mechanism of human ZP proteins.

Identification of an Amyloid Fibril Forming Segment of Human Pmel17 Repeat Domain (RPT Domain)

Pmel17 is the major component of functional amyloid fibrils that have an important role during pigment deposition. Pmel17 polymerization is promoted within the mildly acidic conditions of melanosomes, organelles located in pigment‐specific cells. A repeat domain (RPT domain) of Pmel17, rich in glutamic acid residues has been extensively associated with the formation of the fibrous matrix. Here, we examine the RPT domain of human Pmel17 in order to provide information on this mechanism. Specifically, we have identified an aggregation‐prone peptide segment (405VSIVVLSGT413), close to the C‐terminal part of the RPT domain. Experimental results utilizing electron microscopy, X‐ray fiber diffraction, Congo red staining and ATR FT‐IR spectroscopy indicate that this peptide segment self‐assembles forming fibrils with evident amyloidogenic properties. Conclusively, our results demonstrate that the 405VSIVVLSGT413 peptide segment possibly has an essential role in RPT domain fibrillogenesis.

A β-solenoid model of the Pmel17 repeat domain: insights to the formation of functional amyloid fibrils.

Pmel17 is a multidomain protein involved in biosynthesis of melanin. This process is facilitated by the formation of Pmel17 amyloid fibrils that serve as a scaffold, important for pigment deposition in melanosomes. A specific luminal domain of human Pmel17, containing 10 tandem imperfect repeats, designated as repeat domain (RPT), forms amyloid fibrils in a pH-controlled mechanism in vitro and has been proposed to be essential for the formation of the fibrillar matrix. Currently, no three-dimensional structure has been resolved for the RPT domain of Pmel17. Here, we examine the structure of the RPT domain by performing sequence threading. The resulting model was subjected to energy minimization and validated through extensive molecular dynamics simulations. Structural analysis indicated that the RPT model exhibits several distinct properties of β-solenoid structures, which have been proposed to be polymerizing components of amyloid fibrils. The derived model is stabilized by an extensive network of hydrogen bonds generated by stacking of highly conserved polar residues of the RPT domain. Furthermore, the key role of invariant glutamate residues is proposed, supporting a pH-dependent mechanism for RPT domain assembly. Conclusively, our work attempts to provide structural insights into the RPT domain structure and to elucidate its contribution to Pmel17 amyloid fibril formation.

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.