The silkmoth eggshell as a natural amyloid shield for the safe development of insect oocyte and embryo: insights from studies of silkmoth chorion protein peptide-analogues of the B family

Silkmoth chorion is the major component of the silkmoth eggshell. The proteins that constitute more than 95% of its dry mass have remarkable mechanical and physicochemical properties forming a protective natural shield for the oocyte and the developing embryo from a wide range of environmental hazards. Peptide-analogues of the central conservative domain of the two major families of silkmoth chorion proteins, the A's and the B's, form amyloid fibrils under a variety of conditions, which prompted us to propose, 10 years ago, that silkmoth chorion is an amyloid with protective properties. Following our finding, a number of studies verified the existence of several functional amyloids. In this study, we designed, synthesized and studied two peptide-analogues of the central conservative domain of the B family of silkmoth chorion proteins, and we present experimental results, which show: (a) that the amyloidogenic properties of silkmoth chorion peptides are encoded into the tandemly repeating hexapeptides comprising the central domain of silkmoth chorion proteins, confirming our previous findings from peptide analogues of the A family of chorion proteins, and, (b) they suggest how silkmoth chorion proteins of the B family self-assemble in vivo, for the formation of the helicoidal architecture of silkmoth chorion.     

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.     

Structural models of the evolutionarily conservative central domain of silk-moth chorion proteins

Silk-moth chorion proteins belong to a small number of families: A, B, C, Hc-A and Hc-B. The central domain is an evolutionarily conservative region in each family, of variable length and composition between families. This domain shows clear 6-fold periodicities for various amino acid residues, e.g. glycine. The periodicities, together with the well-documented prevalence of beta-sheet and beta-turn secondary structure of chorion proteins, strongly support a structural model in which four-residue beta-strands alternate with beta-turns, forming a compact antiparallel, probably twisted beta-sheet. Conformational analysis, aided by interactive graphics refinement and recent experimental findings, further suggest that this structure consists of beta-strands, alternating with I' and II' beta-turns, and apparently forms the basis for the molecular and supramolecular assembly of chorion.     

Amyloid fibril formation propensity is inherent into the hexapeptide tandemly repeating sequence of the central domain of silkmoth chorion proteins of the A-family

Peptide-analogues of the A and B families of silkmoth chorion proteins form amyloid fibrils under a variety of conditions [Iconomidou, V.A., Vriend, G. Hamodrakas, S.J. 2000. Amyloids protect the silkmoth oocyte and embryo. FEBS Lett. 479, 141-145; Iconomidou,V.A., Chryssikos, G.D.,Gionis, V., Vriend, G., Hoenger, A., Hamodrakas, S.J., 2001. Amyloid-like fibrils from an 18-residue peptide-analogue of a part of the central domain of the B-family of silkmoth chorion protein. FEBS Lett. 499, 268-273; Hamodrakas, S.J. Hoenger, A., Iconomidou, V. A., 2004 . Amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues via a liquid crystalline intermediate phase. J. Struct. Biol. 145, 226-235.], which led us to propose that silkmoth chorion is a natural protective amyloid. In this study, we designed and synthesized two mutant peptide-analogues of the central conservative domain of the A family: (a) one, cA_m1, with a length half of that of the central domain of the A family, which folds and self-assembles, in various conditions, into amyloid fibrils very similar in properties and structure to the fibrils formed by the cA peptide, which corresponds to the entire length of the A family central domain [Iconomidou, V.A., Vriend, G. Hamodrakas, S.J. 2000. Amyloids protect the silkmoth oocyte and embryo. FEBS Lett. 479, 141-145.], in full support of our previous proposal, (b) the second, cA_m2, differing from cA_m1 at three positions, where three glutamates have replaced two valines and one alanine residues, does not form amyloid fibrils in any conditions. It appears that (a) the amyloidogenic properties of silkmoth chorion peptides are encoded into the tandemly repeating hexapeptides comprising the central domain of silkmoth chorion proteins, and, that (b) suitable mutations, properly and carefully designed, greatly affect the strong amyloidogenic properties inherent in certain aminoacid sequences and may inhibit amyloid formation.     

Amino acid periodicities and their structural implications for the evolutionarily conservative central domain of some silkmoth chorion proteins

The central domain is an evolutionarily conservative region that is invariant in length in the A and Hc-A families of silkmoth chorion proteins. This domain shows strong sixfold periodicities for various amino acid residues, such as glycine and large non-polar residues. The periodicities and their phase relationships, together with the documented prevalence of beta-sheets and beta-turns in the chorion, strongly support a secondary structure model in which short (4-residue) beta-sheet strands alternate with beta-turns, forming a compact antiparallel, probably twisted beta-sheet. This structure should be important for the establishment of higher order structure in the chorion.     

Amyloid-like fibrils from an 18-residue peptide analogue of a part of the central domain of the B-family of silkmoth chorion proteins.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties, and protect the oocyte and the developing embryo from the environment. We present data from negative staining, Congo red binding, X-ray diffraction, Fourier transform-Raman, attenuated total reflectance infrared spectroscopy and modelling studies of a synthetic peptide analogue of a part of the central domain of the B family of silkmoth chorion proteins, indicating that this peptide folds and self-assembles, forming amyloid-like fibrils. These results support further our proposal, based on experimental data from a synthetic peptide analogue of the central domain of the A family of chorion proteins, that silkmoth chorion is a natural, protective amyloid [Iconomidou et al., FEBS Lett. 479 (2000) 141-145].     

Antibacterial activities and conformations of bovine beta-defensin BNBD-12 and analogs:structural and disulfide bridge requirements for activity

Structure and biological activities of synthetic peptides corresponding to bovine neutrophil beta-defensin BNBD-12, GPLSC(1)GRNGGVC(2)IPIRC(3) PVPMRQIGTC(4) FGRPVKC(5) C(6)RSW with disulfide connectivities C(1)-C(5), C(2)-C(4) and C(3)-C(6) and its variants with one, two and three disulfide bridges have been investigated. Selective protection of cysteine thiols was necessary in the four and six cysteine containing peptides for the formation of disulfide connectivities as observed in BNBD-12. Circular dichroism (CD) spectra indicate that in aqueous medium, only a small fraction of molecules populate turn-like conformations. In the presence of micelles and lipid vesicles, the single, two and three disulfide containing peptides adopt beta-hairpin or beta-sheet structures. Antibacterial activity was observed for all the peptides, irrespective of the number of disulfide bridges or how they were connected. Our results suggest that a rigid beta-sheet structure or the presence of three disulfide bridges does not appear to be stringent requirements for antibacterial activity in beta-defensins.     

Structural and functional features of Drosophila chorion proteins s36 and s38 from analysis of primary structure and infrared spectroscopy

Amino acid composition, Fourier transform analysis and secondary structure prediction methods strongly support a tripartite structure for Drosophila chorion proteins s36 and s38. Each protein consists of a central domain and two flanking 'arms'. The central domain contains tandemly repetitive peptides, which apparently generate a secondary structure of beta-sheet strands alternating with beta-turns, most probably, forming a twisted beta-pleated sheet or beta-barrel. The central domains of s36 and s38 share similarities, but they are recognizably different. The flanking 'arms', with different primary and secondary structure features, presumably serve protein-specific functions. The possible roles of the protein domains for the establishment of higher order structure in Drosophila chorion and the possible function of the molecules are discussed. The predicted secondary structure of Drosophila chorion proteins s36 and s38 is supported by experimental information obtained from Fourier transform infrared spectroscopic studies of Drosophila chorions.     

Purification and structural characterization of the central hydrophobic domain of oleosin

The oil bodies of rapeseeds contain a triacylglycerol matrix surrounded by a monolayer of phospholipids embedded with abundant structural alkaline proteins termed oleosins and some other minor proteins. Oleosins are unusual proteins because they contain a 70-80-residue uninterrupted nonpolar domain flanked by relatively polar C- and N-terminal domains. Although the hydrophilic N-terminal domain had been studied, the structural feature of the central hydrophobic domain remains unclear due to its high hydrophobicity. In the present study, we reported the generation, purification, and characterization of a 9-kDa central hydrophobic domain from rapeseed oleosin (19 kDa). The 9-kDa central hydrophobic domain was produced by selectively degrading the N and C termini with enzymes and then purifying the digest by SDS-PAGE and electroelution. We have also reconstituted the central domain into liposomes and synthetic oil bodies to determine the secondary structure of the domain using CD and Fourier transform infrared (FTIR) spectroscopy. The spectra obtained from CD and FTIR were analyzed with reference to structural information of the N-terminal domain and the full-length rapeseed oleosin. Both CD and FTIR analysis revealed that 50-63% of the domain was composed of beta-sheet structure. Detailed analysis of the FTIR spectra indicated that 80% of the beta-sheet structure, present in the central domain, was arranged in parallel to the intermolecular beta-sheet structure. Therefore, interactions between adjacent oleosin proteins would give rise to a stable beta-sheet structure that would extend around the surface of the seed oil bodies stabilizing them in emulsion systems. The strategies used in our present study are significant in that it could be generally used to study difficult proteins with different independent structural domains, especially with long hydrophobic domains.     

Amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues via a liquid-crystalline intermediate phase

Chorion, the major component of silkmoth eggshell, consists of the A and B classes of low-molecular weight structural proteins. Chorion protects the oocyte and the developing embryo from environmental hazards and this is due to the extraordinary physical and chemical properties of its constituent proteins. We have shown previously [FEBS Lett. 479 (2000) 141; 499 (2001) 268] that peptide-analogues of the A and B classes of chorion proteins form amyloid fibrils under a variety of conditions, which led us to propose that silkmoth chorion is a natural, protective amyloid. In this work, we present data showing conclusively that, the first main step of amyloid-like fibrillogenesis of chorion peptides is the formation of nuclei of liquid crystalline nature, which is reminiscent of spider-silk formation. We show that these liquid-crystalline nuclei (spherulites) 'collapse'/deteriorate to form amyloid fibrils in a spectacular manner, important, it seems, for chorion morphogenesis and amyloid fibrillogenesis in general. The molecular 'switch' causing this spectacular transformation is, most probably, a conformational transition to the structure of chorion peptides, from a left-handed parallel beta-helix to an antiparallel beta-pleated sheet. Apparently, these peptides were suitably designed to play this role, after millions of years of molecular evolution.     

Amyloids protect the silkmoth oocyte and embryo

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of proteins that have remarkable mechanical and chemical properties protecting the oocyte and the developing embryo from a wide range of environmental hazards. We present data from electron microscopy (negative staining and shadowing), X-ray diffraction and modeling studies of synthetic peptide analogues of silkmoth chorion proteins indicating that chorion is a natural amyloid. The folding and self-assembly models of chorion peptides strongly support the beta-sheet helix model of amyloid fibrils proposed recently by Blake and Serpell [Structure 4 (1996) 989-998].     

Computational studies on prion proteins: effect of Ala(117)-->Val mutation

Molecular dynamics calculations demonstrated the conformational change in the prion protein due to Ala(117)-->Val mutation, which is related to Gerstmann-Str?ussler-Sheinker disease, one of the familial prion diseases. Three kinds of model structures of human and mouse prion proteins were examined: (model 1) nuclear magnetic resonance structures of human prion protein HuPrP (125-228) and mouse prion protein MoPrP (124-224), each having a globular domain consisting of three alpha-helices and an antiparallel beta-sheet; (model 2) extra peptides including Ala(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1; and (model 3) extra peptides including Val(117) (109-124 in HuPrP and 109-123 in MoPrP) plus the nuclear magnetic resonance structures of model 1. The results of molecular dynamics calculations indicated that the globular domains of models 1 and 2 were stable and that the extra peptide in model 2 tended to form a new alpha-helix. On the other hand, the globular domain of model 3 was unstable, and the beta-sheet region increased especially in HuPrP.     

Design and NMR conformational study of a beta-sheet peptide based on Betanova and WW domains

A good approach to test our current knowledge on formation of protein beta-sheets is de novo protein design. To obtain a three-stranded beta-sheet mini-protein, we have built a series of chimeric peptides by taking as a template a previously designed beta-sheet peptide, Betanova-LLM, and incorporating N- and/or C-terminal extensions taken from WW domains, the smallest natural beta-sheet domain that is stable in absence of disulfide bridges. Some Betanova-LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt beta-sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest beta-sheet population according to the CD spectra, named 3SBWW-2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW-2 adopts a well defined three-stranded antiparallel beta-sheet structure with a disordered C-terminal tail. To discern between the contributions to beta-sheet stability of strand residues and the C-terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW-2 but lacking the C-terminal extension, named Betanova-LYYL, was also investigated. beta-Sheet stability in these two peptides, in the parent Betanova-LLM and in WW-P, a prototype WW domain, decreased in the order WW-P > 3SBWW-2 > Betanova-LYYL > Betanova-LLM. Conclusions about the contributions to beta-sheet stability were drawn by comparing structural properties of these four peptides.     

Conformational studies of Aib-rich peptides containing lactam-bridged side chains: evidence of 3(10)-helix formation

Aib-rich side-chain lactam-bridged oligomers Ac-(Glu-Aib-Aib-Lys)n-Ala-OH with n = 1,2,3 were designed and synthesized as putative models of the 3(10)-helix. The lactam bridge between the side chains of L-Glu and L-Lys in (i)--(i + 3) positions was introduced in order to enhance the structural preference toward the right-handed 3(10)-helix. The conformational properties of the three peptides were studied in trifluoroethanol (TFE) solution by CD, NMR, and computer simulations. The structural information was derived mainly from the analysis of nuclear Overhauser effect spectroscopy spectra. The presence of alpha H(i)-HN(i + 2) and of alpha H(i)-HN(i + 3) connectivities and the absence of alpha H(i)-HN(i + 4) connectivities indicate that these peptides fold into a 3(10)-helix rather than into an alpha-helix. Based on these conformational features, stereospecific assignment of the Aib methyl groups was possible. The results of such experiments and of the subsequent distance geometry and restrained molecular dynamics simulations reveal a marked preference of these peptides for 3(10)-helix. The CD spectra of these peptides indicate that the helix content increases upon chain elongation. The CD spectrum of the trimer is characterized by a negative band at 200 nm and by a weak positive band around 220 nm. The CD spectrum in TFE is different from that observed in aqueous solution in the presence of SDS micelles, reported in our previous work, and from those reported by a different research group for 3(10)-helical peptides. A possible reason for these differences could rest in the presence of different equilibria of the conformer populations of the various peptides in different solvent systems.     

On (GGLGY) synthetic repeating sequences of lamprin and analogous sequences.

The repetitive sequence GGLGY was found in lamprin, the most important matrix protein of lamprey annular cartilage by Keeley and co-workers. Similar sequences appear also in other proteins, i.e. elastin, spidroin, spider minor ampullate silk proteins, in matrix proteins of the chorion or egg shell membrane of insects and others. We synthesized (GGLGY)n, n=1, 2, 6, because the sequence is repeated six times in the aggregated protein. The peptides were studied both in solution and in the solid state. Because the CD spectra were dominated by aromatic contribution, we synthesized GGLGF and GGLGA in order to carefully interpret the CD spectra. The conformational analysis suggests that all synthetic peptides do adopt the same secondary structure. In solution the peptides present a flexible conformation with a significant amount of PPII structure. In the solid state PPII, beta-pleated-sheets and beta-turns possibly co-exist.     

Structural transitions involved in a novel amyloid-like beta-sheet assemblage of tripeptide derivatives

Self-assembly of two tripeptide derivatives containing three nonpolar isoleucine moieties and polar oxyethylene groups are studied in methanol. Peptide A [CH3(OCH2CH2)3OCH2CO(Ile)3OCH3] and peptide B [CH3(OCH2CH2)3OCH2CO(Ile)3NH (CH2CH2O)3CH3] take a mixture of unordered and helical conformation at low concentration (8.5 x 10(-4) M). However, at high concentration (2 x 10(-3) M), both the peptide showed significant increase in the helical conformation. An interesting conformational transition of peptides A and B at various methanol contents was observed in the solvated films of these compounds by spectroscopic methods like the far-uv circular dichroism and Fourier transform infrared (FT-IR) techniques. Peptide B, which contains more polar oxyethylene groups than A, showed a highly cooperative conformational transition when the methanol content was decreased. This transition was characterized by a large increase of beta-sheet, retaining a alpha-helical contribution. Peptide A showed a conformational transition resulting in a beta-sheet in the aggregated state. From the CD spectra, the ratio in the ellipticity indicates that peptide B forms twisted antiparallel beta-sheet conformation, whereas peptide A takes a parallel beta-sheet conformation. The results obtained in this work indicates the role of polar derivatization on the conformational preference of peptides having similar sequence.     

Effect of membrane mimicking environment on the conformation of a pore-forming (xSxG)6 peptide

The mechanism of membrane interaction by beta-sheet peptides is important to understand fundamental principles of folding of beta-barrel proteins and various beta-amyloid proteins. Here, we examined the conformational characteristics of a porin-like channel forming (xSxG)(6) peptide in solution and membrane-mimicking environments (CD and ATR-IR) to understand the structural changes of the peptide during membrane association and channel formation. A comparison of the peptide conformations in different microenvironments showed that beta-sheet formation is enhanced in membrane-mimicking liposomes and SDS-micelles. The lipid-induced beta-sheet formation was confirmed by the formation of a characteristic beta-sheet structure on mixing a methanolic solution of the peptide (partially folded) with preformed liposomes. The amphipathicity of the peptide; increased hydrogen bonding, hydrophilicity, and reduction in dimensionality of the membrane surface; membrane-peptide interaction-forces; and presence of flexible glycines might facilitate beta-sheet formation in membranes. Though the CD spectra of both the peptide-bound and peptide-incorporated lipids are reminiscent of a beta-sheet structure, a significant variation in the peak positions of the two beta-sheet structures was noticed. The channel characteristics of (xSxG)(6) in the presence of low ionic strength solutions of NEt(3)BzCl and glucosammonium chloride are comparable to those reported under high ionic strength solutions. Altogether the data suggest that the channel formation by (xSxG)(6) proceeds via beta-sheet aggregate formation at the membrane surface, beta-sheet insertion, and rearrangement into a beta-barrel-like structure. The beta-barrel-like channel formation most likely arises from a sequence similarity to beta-barrel porins whereas the lipid-induced beta-sheet formation is governed by the above-mentioned factors.     

LepChorionDB,a database of Lepidopteran chorion proteins and a set of tools useful for the identification of chorion proteins in Lepidopteran proteomes

Chorion proteins of Lepidoptera have a tripartite structure, which consists of a central domain and two, more variable, flanking arms. The central domain is highly conserved and it is used for the classification of chorion proteins into two major classes, A and B. Annotated and unreviewed Lepidopteran chorion protein sequences are available in various databases. A database, named LepChorionDB, was constructed by searching 5 different protein databases using class A and B central domain-specific profile Hidden Markov Models (pHMMs), developed in this work. A total of 413 Lepidopteran chorion proteins from 9 moths and 1 butterfly species were retrieved. These data were enriched and organised in order to populate LepChorionDB, the first relational database, available on the web, containing Lepidopteran chorion proteins grouped in A and B classes. LepChorionDB may provide insights in future functional and evolutionary studies of Lepidopteran chorion proteins and thus, it will be a useful tool for the Lepidopteran scientific community and Lepidopteran genome annotators, since it also provides access to the two pHMMs developed in this work, which may be used to discriminate A and B class chorion proteins. LepChorionDB is freely available at http://bioinformatics.biol.uoa.gr/LepChorionDB.     

Antibacterial activities and conformations of synthetic alpha-defensin HNP-1 and analogs with one, two and three disulfide bridges.

Structure and biological activities of synthetic peptides corresponding to human alpha-defensin HNP-1, AC1YC2RIPAC3IAGERRYGTC4IYQGRLWAFC5C6 with the S-S connectivities: C1-C6, C2-C4, C3-C5, and its variants with one, two and three disulfide bridges were investigated. Oxidation of synthetic, reduced HNP-1 yielded a peptide with S-S connectivities C1-C3, C2-C4 and C5-C6, and not with the S-S linkages as in naturally occurring HNP-1. Selective protection of cysteine sulfhydryls was necessary for the formation of S-S bridges as in native HNP-1. Likewise, oxidation of peptide encompassing the segment from C2 to C5, resulted in the S-S linkages C2-C3 and C4-C5 instead of the expected linkage C2-C4 and C3-C5. Antibacterial activities were observed for all peptides, irrespective of how the S-S bridges were linked. Linear peptides without S-S bridges were inactive. Circular dichroism (CD) spectra suggest that peptides constrained by one and two S-S bridges do not form rigid beta-sheet structures in an aqueous environment. The spectrum of HNP-1 in an aqueous environment suggests the presence of a beta-hairpin conformation. In the presence of lipid vesicles, the S-S constrained peptides tend to adopt a beta-structure. Although the S-S connectivities observed in HNP-1 may be necessary for other physiological activities, such as chemotaxis, they are clearly not essential for antibacterial activity.     

Model peptide studies of sequence repeats derived from the intracrystalline biomineralization protein, SM50. II. Pro,Asn-rich tandem repeats

In the biomineralization process, a number of Pro-rich proteins participate in the formation of three-dimensional supramolecular structures. One such protein superfamily, the Pro,Gly-rich sea urchin intracrystalline spicule matrix proteins, form protein-protein supramolecular assemblies that modify the microstructure of the inorganic mineral phase (calcite) within embryonic sea urchin spicules and adult sea urchin spines. These proteins represent a useful model for understanding Pro sequence usage and the resulting generation of extended or "open" structures for protein-protein and/or protein-crystal recognition. In the sea urchin spicule matrix protein, SM50 (Strongylocentrotus purpuratus), there exists an unusual 20-residue Pro,Asn-containing repeat, &bond;PNNPNNPNPNNPNNPNNPNPbond which links the upstream 15-residue C-terminal domain and the downstream 211-residue beta-spiral repeat domain. To define the structural preferences of this 20-residue repeat, we created a 20-residue N- and C-terminal "capped" peptidomimetic of this sequence. Using far-uv CD dichroism, CH(alpha) and alpha-(15)N conformational shifts, (3)J(NH-CHalpha) coupling constants, sequential d(NN(i, i + 1)) rotating frame nuclear Overhauser effect connectivities, d(alphaN(i, i + 1))/d(NN(i, i + 1)) intensity ratios, amide temperature shift coefficients, amide solvent exchange, and simulated annealing refinement protocols, we have determined that this 20-residue repeat motif adopts an extended "twist" structure consisting of turn- and coil-like regions. These findings are consistent with previous studies, which have shown that Pro-rich tandem repeats adopt extended, flexible structures in solution. We hypothesize that this 20-residue repeat may fulfill the role of a mineral-binding domain, a protein-protein docking domain, or as an internal "molecular spacer" for the SM50 protein during spicule biocomposite formation.