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

Hidden Aggregation Hot-Spots on Human Apolipoprotein E: A Structural Study

Human apolipoprotein E (apoE) is a major component of lipoprotein particles, and under physiological conditions, is involved in plasma cholesterol transport. Human apolipoprotein E found in three isoforms (E2; E3; E4) is a member of a family of apolipoproteins that under pathological conditions are detected in extracellular amyloid depositions in several amyloidoses. Interestingly, the lipid-free apoE form has been shown to be co-localized with the amyloidogenic Aβ peptide in amyloid plaques in Alzheimer’s disease, whereas in particular, the apoE4 isoform is a crucial risk factor for late-onset Alzheimer’s disease. Evidence at the experimental level proves that apoE self-assembles into amyloid fibrilsin vitro, although the misfolding mechanism has not been clarified yet. Here, we explored the mechanistic insights of apoE misfolding by testing short apoE stretches predicted as amyloidogenic determinants by AMYLPRED, and we computationally investigated the dynamics of apoE and an apoE−Αβ complex. Our in vitro biophysical results prove that apoE peptide−analogues may act as the driving force needed to trigger apoE aggregation and are supported by the computational apoE outcome. Additional computational work concerning the apoE−Αβ complex also designates apoE amyloidogenic regions as important binding sites for oligomeric Αβ; taking an important step forward in the field of Alzheimer’s anti-aggregation drug development

X-ray diffraction patterns produced from oriented fibres of mature fibril suspensions.

<p>The mature fibrils have derived from: (a) ZPH_A peptide, (b) ZPH_G peptide, (c) a mixture of ZPH_A & ZPH_G peptides. The meridian, M (direction parallel to the fibre axis, F) is vertical and the equator, E, is horizontal in this display. All X-ray diffraction patterns are clearly “cross-β” patterns <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Geddes1" target="_blank">[71]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Jahn1" target="_blank">[73]</a>. (a) An intense meridional 4.7 Å reflection corresponds to the spacing of successive hydrogen bonded β-strands, perpendicular to the fiber axis, whereas the 9.1 Å reflection on the equator is attributed to the packing distance of β-sheets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 4). The sheets are packed parallel to the fiber axis. (b) The X-ray diffraction pattern of the ZPH_G peptide also exhibits similar reflections that indicate the presence of a “cross-β” conformation. The structural repeat of 4.7 Å corresponds to the spacing of successive β-strands arranged perpendicular to the fiber axis, while the 12.4 Å spacing on the equator, corresponds to the packing distance of consecutive β-sheet parallel to the fibre axis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 5). (c) The X-ray pattern produced from the mixture of the ZPH_A & ZPH_G peptide fibril suspensions is clearly a combination of the diffraction patterns produced by the individual fibers formed from the ZPH_A and ZPH_G peptide's fibril suspensions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t001" target="_blank">Table 1</a>, column 6). The 2.4 Å, 3.8 Å, 7.1 Å and 9.1 Å reflections are due to the presence of fibrils formed by the ZPH_A peptide, whereas the 12.4 Å reflection is produced by fibrils formed by the ZPH_G peptide (corresponding to β-sheet packing distance). The intense 4.7 Å reflection has contributions from both fibril populations and this is in agreement with the EM photograph of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-g002" target="_blank">Fig. 2c</a>.</p

Sequence alignment of the ZP-N domain of mouse ZP3 and human ZP1 proteins.

<p>The crystallographically determined secondary structure elements are depicted below the sequences: Arrows and helices represent observed beta-strands (named consecutively A to G) and alpha helices, respectively. The invariant cysteine residues connected by disulfide bonds (dotted lines) are also seen. Peptides ATVQCF and FQLHVRC, corresponding to the beta strands A and G of human ZP1, respectively, predicted by AMYLPRED <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Frousios1" target="_blank">[43]</a> as ‘aggregation-prone’ stretches, are enclosed in boxes (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#s2" target="_blank">Materials and methods</a>’).</p

Electron micrographs of amyloid-like fibrils, negatively stained with 1% uranyl acetate.

<p>Amyloid-like fibrils were derived by self-assembly, from a 10 mg ml⁻¹ solution of peptides ZPH_A (a) and ZPH_G (b) in distilled water, pH 5.5. A solution of a mixture of the ZPH_A and ZPH_G peptides (in 1∶1 ratio, in distilled water, pH 5.5, concentration 5 mg ml⁻¹ per peptide) also revealed the formation of amyloid-like fibrils, after an incubation period of ca. one week (c). (a) They are unbranched and of undetermined length, approximately 100–120 Å in diameter and have a double helical structure. A pair of protofilaments each 40–50 Å in diameter wrap around each other, with intervening stain between them, thus forming double-helical fibrils (arrows). Bar 500 nm. (b) Protofilaments interact laterally, forming ribbons and, eventually, gels. The fibrils formed exhibit a characteristic for amyloid-like fibrils polymorphism <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Kodali1" target="_blank">[67]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone.0073258-Pedersen1" target="_blank">[69]</a>. Bar 200 nm. (c) Two different types (populations) of fibrils are apparent, due to “self-aggregation” of each peptide (double and single arrows, respectively), which are similar to those viewed separately by the ZPH_A and ZPH_G peptide solutions in (a) and (b) above. Bar 500 nm.</p

Photomicrographs of peptide fibrils stained with Congo red.

<p>Fibrils have derived from: ZPH_A (a–b), ZPH_G (c–d) and ZPH_A & ZPH_G mixture (e–f) peptides, respectively (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#s2" target="_blank">Materials and methods</a>’). Solutions of these peptides, after ca. one (1) week incubation, produced fibrils, which were then stained with Congo red. The typical for amyloid fibrils apple-green birefringence is clearly seen, under crossed polars. (a,c,e) Bright field illumination, (b,d,f) Crossed polars Bar 400 µm.</p

Bands observed in the IR spectrum of a hydrated film produced from a suspension of fibrils produced by ZPH_A peptide, ZPH_G peptide and from a mixture of both peptides, dissolved in equal (1∶1) amounts and their tentative assignments (Fig. 5).

<p>Bands observed in the IR spectrum of a hydrated film produced from a suspension of fibrils produced by ZPH_A peptide, ZPH_G peptide and from a mixture of both peptides, dissolved in equal (1∶1) amounts and their tentative assignments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-g005" target="_blank">Fig. 5</a>).</p

ATR FT-IR (1100–1800 cm⁻¹) spectra obtained from thin hydrated-films containing mature amyloid-like fibrils.

<p>These thin hydrated-films, formed from (a) ZPH_A, (b) ZPH_G and (c) the mixture of ZPH_A & ZPH_G peptides, were cast on flat stainless-steel plates coated with an ultra thin hydrophobic layer (see ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#s2" target="_blank">Materials and methods</a>’). Second derivative spectra are also included and were used for the exact identification of the band maxima and their tentative assignments. All resulting spectra are indicative of the preponderance of an antiparallel β-sheet secondary structure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073258#pone-0073258-t002" target="_blank">Table 2</a>).</p

Arabidopsis thaliana Plant Natriuretic Peptide Active Domain Forms Amyloid-like Fibrils in a pH-Dependent Manner

Plant natriuretic peptides (PNPs) are hormones that have been extracted from many different species, with the Arabidopsis thaliana PNP (AtPNP-A) being the most studied among them. AtPNP-A is a signaling molecule that consists of 130 residues and is secreted into the apoplast, under conditions of biotic or abiotic stress. AtPNP-A has distant sequence homology with human ANP, a protein that forms amyloid fibrils in vivo. In this work, we investigated the amyloidogenic properties of a 34-residue-long peptide, located within the AtPNP-A sequence, in three different pH conditions, using transmission electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, Congo red and Thioflavin T staining assays. We also utilize bioinformatics tools to study its association with known plant amyloidogenic proteins and other A. thaliana proteins. Our results reveal a new case of a pH-dependent amyloid forming peptide in A. thaliana, with a potential functional role