Chicken Prion Research Articles

How copper ions and membrane environment influence the structure of the human and chicken tandem repeats domain?

Prion proteins (PrPs) from different species have the enormous ability to anchor copper ions. The N-terminal domain of human prion protein (hPrP) contains four tandem repeats of the –PHGGGWGQ– octapeptide sequence. This octarepeat domain can bind up to four Cu2+ ions. Similarly to hPrP, chicken prion protein (chPrP) is able to interact with Cu2+ through the tandem hexapeptide -HNPGYP- region (residues 53–94). In this work, we focused on the human octapeptide repeat (human Octa4, hPrP60–91) (Ac-PHGGGWGQPHGGGWGQPHGGGWGQPHGGGWGQ-NH2) and chicken hexapeptide repeat (chicken Hexa4, chPrP54–77) (Ac-HNPGYPHNPGYPHNPGYPHNPGYP-NH2) prion protein fragments. Due to the fact that PrP is a membrane-anchored glycoprotein and its unstructured and flexible N-terminal domain may interact with the lipid bilayer, our studies were carried out in presence of the surfactant sodium dodecyl sulfate (SDS) mimicking the membrane environment in vitro. The main objective of this work was to understand the effects of copper ion on the structural rearrangements of the human and chicken N-terminal repeat domain. The obtained results provide a fundamental first step in describing the thermodynamic (potentiometric titrations) and structural properties of Cu(II) binding (UV–Vis, NMR, CD spectroscopy) to both human Octa4 and chicken Hexa4 repeats in both a DMSO/water and SDS micelle environment. Interestingly, in SDS environment, both ligands indicate different copper coordination modes, which results of the conformational changes in micelle environment. Our results strongly support that copper binding mode strongly depends on the protein backbone structure. Moreover, we focused on previously obtained results for amyloidogenic human and chicken fragments in membrane mimicking environment.

The first report of genetic variations in the chicken prion protein gene

ABSTRACTAbnormal structural changes of the prion protein (PrP) are the cause of prion disease in a wide range of mammals. However, spontaneous infected cases have not been reported in chicken. Genetic variations of the prion protein gene (PRNP) may impact susceptibility to prion disease but have not been investigated thus far. Because an investigation of the chicken PRNP can improve the understanding of characteristics related to resistance to prion disease, research on the chicken PRNP is highly desirable. In this study, we investigated the genetic characteristics of the chicken PRNP gene. For this, we performed direct sequencing in 106 Dekalb White chickens and analyzed the genotype and allele frequencies of chicken PRNP gene. We found two insertion and deletion polymorphisms in the chicken PRNP: c.163_180delAACCCAGGGTACCCCCAT and c.268_269insC. The former is a U2 hexapeptide deletion polymorphism. Of the 106 samples, 13 (12.26%) were insertion homozygotes, 89 (83.96%) were heterozygotes, and 4 (3.77%) were deletion homozygotes in c.163_180delAACCCAGGGTACCCCCAT. In the c.268_269insC polymorphism, 102 (96.23%) were deletion homozygotes, and 4 (3.77%) were heterozygotes. Insertion homozygotes of c.268_269insC were not detected. Two polymorphisms were in perfect linkage disequilibrium (LD) with a D’ value of 1.0, and three haplotypes were identified. Furthermore, PROVEAN evaluates 163_180delAACCCAGGGTACCCCCAT as ‘deleterious’ with a score of – 13.173. Furthermore, single nucleotide polymorphisms (SNPs) in the open reading frame (ORF) of the PRNP gene were not found in the chicken. To the best of our knowledge, this was the first report on the genetic variations of the chicken PRNP gene.

A multispecific monoclonal antibody G2 recognizes at least three completely different epitope sequences with high affinity.

A monoclonal antibody (mAb) G2 possesses an unusual characteristic of reacting with at least three proteins (ATP6V1C1, SEPT3, and C6H10orf76) other than its original antigen, chicken prion protein (ChPrP). The epitopes on ChPrP and ATP6V1C1 have been identified previously. In this study, we identified the epitope in the third protein, SEPT3. Interestingly, there was no amino acid sequence similarity among the epitopes on the three proteins. These epitopes had high binding affinities to G2 (KD = ∼10-7 M for monovalent binding and KD = ∼10-9 M for divalent binding), as determined using a SPR biosensor. This is the first report on a three-in-one mAb recognizing completely different epitope sequences with high affinity. Additionally, competitive ELISA indicated that the binding sites on G2, specific for the three different epitopes, overlapped, suggesting that the antigen-binding site may be flexible in the free form and capable of adapting to at least three different conformations to enable interactions with three different antigens.

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments.

The effect of a membrane-mimicking environment on the interactions of Cu2+with an amyloidogenic fragment of chicken prion protein

Prion proteins (PrP) from different species have the ability to tightly bind Cu2+ ions. Copper coordination sites are located in the disordered and flexible N-terminal region which contains several His anchoring sites. Among them, two His residues are found in the so called amyloidogenic PrP region which is believed to play a key role in the process leading to oligomer and fibril formation. Both chicken and human amyloidogenic regions have a hydrophobic C-terminal region rich in Ala and Val amino acids. Recent findings revealed that this domain undergoes random coil to α-helix structuring upon interaction with membrane models. This interaction might strongly impact metal binding abilities either in terms of donor sets or affinity. In this study we investigated Cu2+ interaction with an amyloidogenic fragment, chPrP105-140, derived from chicken prion protein (chPrP), in different solution environments. The behavior of the peptide and its metal complexes was analyzed in water and in the presence of negative and positive charged membrane mimicking environments formed by sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium chloride (DTAC) micelles. The metal coordination sphere, the metal binding affinity and stoichiometry were evaluated by combining spectroscopic and potentiometric methods. Finally we compare copper(ii) interactions with human and chicken amyloidogenic fragments. Our results indicate that the chicken amyloidogenic fragment is a stronger copper ligand than the human amyloidogenic fragment.

Downregulation of cellular prion protein inhibited the proliferation and invasion and induced apoptosis of Marek's disease virus-transformed avian T cells

Cellular prion protein (PrPC) is ubiquitously expressed in the cytomembrane of a considerable number of eukaryotic cells. Although several studies have investigated the functions of PrPC in cell proliferation, cell apoptosis, and tumorigenesis of mammals, the correlated functions of chicken PrPC (chPrPC) remain unknown. In this study, stable chPrPC-downregulated Marek's disease (MD) virus-transformed avian T cells (MSB1-SiRNA-3) were established by introducing short interfering RNA (SiRNA) targeting chicken prion protein genes. We found that downregulation of chPrPC inhibits proliferation, invasion, and migration, and induces G1 cell cycle phase arrest and apoptosis of MSB1-SiRNA-3 cells compared with Marek's disease virus-transformed avian T cells (MSB1) and negative control cells. To the best of our knowledge, the present study provides the first evidence supporting the positive correlation between the expression level of chPrPC and the proliferation, migration, and invasion ability of MSB1 cells, but appears to protect MSB1 cells from apoptosis, which suggests it functions in the formation and development of MD tumors. This evidence may contribute to future research into the specific molecular mechanisms of chPrPC in the formation and development of MD tumors.

Identification and characterization of a multispecific monoclonal antibody G2 against chicken prion protein.

We previously generated a monoclonal antibody (mAb), G2, by immunizing mice with Residues 174-247 of the chicken prion protein (ChPrP(C) ). In this study, we found that G2 possessed an extremely unusual characteristic for a mAb; in particular, it could react with at least three proteins other than ChPrP(C) , the original antigenic protein. We immunoscreened a complementary DNA library from chicken brain DNA and found three proteins (SEPT3, ATP6V1C1, and C6H10orf76) that reacts with G2. There were no regions of amino acid sequence similarity between ChPrP(C) and SEPT3, ATP6V1C1, or C6H10orf76. We selected ATP6V1C1 as a representative of the three proteins and identified the epitope within ATP6V1C1 that reacts with G2. The amino acid sequence of the G2 epitope within ATP6V1C1 (Pep8) was not related to the G2 epitope within ChPrP(C) (Pep18mer). However, enzyme-linked immunosorbent assay, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) experiments indicated that these two peptides have similar binding affinity for G2. The apparent KD values of Pep18mer and Pep8 obtained from SPR experiments were 2.9 × 10(-8) and 1.6 × 10(-8) M, respectively. Antibody inhibition test using each peptide indicated that the binding sites of the two different peptides overlapped each other. We observed that these two peptides substantially differed in several binding characteristics. Based on the SPR experiments, the association and dissociation rate constants of Pep18mer were higher than those of Pep8. A clear difference was also observed in ITC experiments. These differences may be explained by G2 adopting different binding conformations and undergoing different binding pathways.

Probing the Residual Structure in Avian Prion Hexarepeats by CD, NMR and MD Techniques

Many proteins perform essential biological functions by means of regions that lacking specific organized structure exist as an ensemble of interconverting transient conformers. The characterization of such regions, including the description of their structural propensities, number of conformations and relative populations can provide useful insights. Prion diseases result from the conversion of a normal glycoprotein into a misfolded pathogenic isoform. The structures of mammal and chicken prion proteins show a similar fold with a globular domain and a flexible N-terminal portion that contains different repeated regions: octarepeats (PHGGGWGQ) in mammals and hexarepeats (PHNPGY) in chickens. The higher number of prolines in the hexarepeat region suggests that this region may retain a significant amount of residual secondary structure. Here, we report the CD, NMR and MD characterization of a peptide (2-HexaPY) composed of two hexarepeats. We combine experimental NMR data and MD to investigate at atomic level its ensemble-averaged structural properties, demonstrating how each residue of both repeats has a different quantified PPII propensity that shows a periodicity along the sequence. This feature explains the absence of cooperativity to stabilize a PPII conformation. Nonetheless, such residual structure can play a role in nucleating local structural transitions as well as modulating intra-molecular or inter-molecular interactions.

Replica Exchange MD Nnvestigation of the Conformational Space of Prion Proteins

Prion proteins are found on the surface of nerve cells. Their function is not fully understood yet, but they are related to the etiology of certain rare deseases, like CJD, GSS, Kuru, FFI etc [1]. Prions exist in a native (PrP) and in a highly infective pathological form (PrPSc – scrapie form). PrPSc proteins can transform native prions into scrapie forms, aggregate and thus lead to cellular death. Experimental insights on the scrapie form suggest a higher fibrilar beta-structure content, in contrast to the mostly globular alpha-helical native form [2]. However, the 3D structure of PrPSc is still unknown. The present study aims at identification of scrapie form candidates, investigating the prion conformational space by means of replica exchange molecular dynamics. Thus, a conformation of a chicken prion protein is constructed with beta-structure content in agreement with the experimental data.

1P050 トリプリオンタンパク質に対する抗体G2の複数の抗原を特異的に認識する性質の特徴付け(01C. 蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

1P050 トリプリオンタンパク質に対する抗体G2の複数の抗原を特異的に認識する性質の特徴付け(01C. 蛋白質:物性,ポスター,日本生物物理学会年会第51回(2013年度))

Membrane Interactions and Conformational Preferences of Human and Avian Prion N-Terminal Tandem Repeats: The Role of Copper(II) Ions, pH, and Membrane Mimicking Environments

The flexible N-terminal domain of the prion protein (PrP(c)) is believed to play a pivotal role in both trafficking of the protein through the cell membrane and its pathogenic conversion into the β sheet-rich scrapie isoform (PrP(sc)). Unlike mammalian PrP(c), avian prion proteins are not known to undergo any pathogenic conformational conversions. Consequently, some critical advances in our understanding of the molecular mechanisms underlying prion pathogenesis are expected from comparative studies of the biophysical properties of the N-terminal domains of the two proteins. The present study addresses the role played by different environmental factors, i.e., copper(II), pH, and membrane-mimicking environments, in assisting the conformational preferences of huPrP60-91 and chPrP53-76, two soluble peptides encompassing the N-terminal copper(II) binding domains of the human and chicken prion proteins, respectively. Moreover, the membrane interactions of huPrP60-91, chPrP53-76, and their copper(II) complexes were evaluated by Trp fluorescence in conjunction with measurements of the variation in thermotropic properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) unilamellar vesicles. Circular dichroism experiments revealed that huPrP60-91 adopts a predominant polyproline II conformation in aqueous solution that is destabilized at basic pH or in the presence of trifluoroethanol (TFE). Unlike anionic sodium dodecyl sulfate (SDS), which seems to stabilize the polyproline II conformation further, zwitterionic dodecylphosphocholine (DPC) micelles do not affect the peptide structure. On the contrary, copper(II) promptly promotes an increase in β-turn-rich structures. Differential scanning calorimetry (DSC) and Trp fluorescence assays carried out on DPPC model membranes after incubation with huPrP60-91 showed a marked tendency of the peptide to slowly penetrate the lipid bilayer with a concomitant conformational transition toward an extended β-sheet-like structure. Such an event, which was ascribed to the hydrophobic Trp side chain residues, was shown to also depend on the level of copper(II) occupancy along the peptide. Conversely, the CD spectra of chPrP53-76 aqueous solutions indicated the presence of a mixture of random-coil/β-turn-like structures whose resulting equilibrium was influenced by SDS and copper(II) addition. Furthermore, chPrP53-76 did not exhibit any tendency to interact with model membranes in either the presence or absence of copper(II). The results reported here provide evidence of the different roles played by environmental factors in affecting the conformation and membrane activity of human and avian prion N-terminal domains.

Copper binding to chicken and human prion protein amylodogenic regions: Differences and similarities revealed by Ni2+ as a diamagnetic probe

Both human (h) and chicken (Ch) prion proteins (PrP) bind copper ions within the so called "tandem repeat" N-terminal region. Outside this region, hPrP possesses two additional copper binding sites, localized at His-96 and His-111 in the so called "amylodogenic" or neurotoxic region (residues 91-126). Also ChPrP possesses a similar region (ChPrP(105-140)) containing two His (His-110 and His-124) and an identical hydrophobic tail of 15 amino acids rich in Ala and Gly. The copper binding abilities within such region of ChPrP were investigated by NMR, CD and potentiometry using Ni(2+) as diamagnetic probe. The formation of diamagnetic metal complexes allowed to monitor the chemical shift and signal intensity variations and to determine the structural and kinetic features of the His-110 and His-124 metal binding sites. Finally a comparison between the hPrP and ChPrP metal binding abilities was performed. We found that the two prion proteins exhibited different copper and nickel preferences with the favoured metal binding sites localized at opposite His: His-110 for ChPrP, and His-111 for hPrP.

Conformational Preferences of the Full Chicken Prion Protein in Solution and Its Differences with Respect to Mammals

We investigate the conformations of the full chicken prion protein (ChPrP1-267) in solution at neutral pH with molecular dynamics simulations. We focus on the persistence of its secondary structure motifs using a recently proposed protein chirality indicator [A. Pietropaolo et al., Proteins 2008, 70, 667-677]. From this, we find a high rigidity of helix 2 (ChPrP178-195) and of the hexarepeat domain, which is turn rich, and a plasticity of the short beta-sheet, consistent with the available NMR structural details. We also determine the extent of solvation for each residue, revealing local minima for such structured regions. These features hint at a possible origin of the high resistance to proteolysis of the avian prion proteins and of its capability in preventing the aggregation in comparison to mammals.

Structural Domains and Main-Chain Flexibility in Prion Proteins

In this study we describe a novel approach to define structural domains and to characterize the local flexibility in both human and chicken prion proteins. The approach we use is based on a comprehensive theory of collective dynamics in proteins that was recently developed. This method determines the essential collective coordinates, which can be found from molecular dynamics trajectories via principal component analysis. Under this particular framework, we are able to identify the domains where atoms move coherently while at the same time to determine the local main-chain flexibility for each residue. We have verified this approach by comparing our results for the predicted dynamic domain systems with the computed main-chain flexibility profiles and the NMR-derived random coil indexes for human and chicken prion proteins. The three sets of data show excellent agreement. Additionally, we demonstrate that the dynamic domains calculated in this fashion provide a highly sensitive measure of protein collective structure and dynamics. Furthermore, such an analysis is capable of revealing structural and dynamic properties of proteins that are inaccessible to the conventional assessment of secondary structure. Using the collective dynamic simulation approach described here along with a high-temperature simulations of unfolding of human prion protein, we have explored whether locations of relatively low stability could be identified where the unfolding process could potentially be facilitated. According to our analysis, the locations of relatively low stability may be associated with the beta-sheet formed by strands S1 and S2 and the adjacent loops, whereas helix HC appears to be a relatively stable part of the protein. We suggest that this kind of structural analysis may provide a useful background for a more quantitative assessment of potential routes of spontaneous misfolding in prion proteins.

Copper(ii) coordination outside the tandem repeat region of an unstructured domain of chicken prion protein

Combined potentiometric, calorimetric and spectroscopic methods were used to investigate the Cu(2+) binding ability and coordination behaviour of some peptide fragments related to the neurotoxic region of chicken Prion Protein. The systems studied were the following protein fragments: chPrP(106-114), chPrP(119-126), chPrP(108-127), chPrP(105-127) and chPrP(105-133).The complex formation always starts around pH 4 with the coordination of an imidazole nitrogen, followed by the deprotonation and binding of amide nitrogens from the peptidic backbone. At neutral pH, the {N(im), 3N(-)} binding mode is the preferred one. The amide nitrogens participating in the binding to the Cu(2+) ion derive from residues from the N-terminus side, with the formation of a six-membered chelate ring with the imidazolic side chain.Comparison of thermodynamic data for the two histydyl binding domains (around His-110 and His-124), clearly indicates that the closest to the hexarepeat domain (His-110) has the highest ability to bind Cu(2+) ions, although both of them have the same coordination mode. Conversely, in the case of the human neurotoxic peptide region, between the two binding sites, located at His-96 and His-111, the farthest from the tandem repeat region is the strongest one. Finally, thermodynamic data show that chicken peptide is a distinctly better ligand for coordination of copper ions with respect to the human fragment.

Prion Proteins Leading to Neurodegeneration

Prion diseases are fatal neurodegenerative disorders related to the conformational alteration of the prion protein (PrP C) into a pathogenic and protease-resistant isoform PrP(Sc). PrP(C) is a cell surface glycoprotein expressed mainly in the central nervous system and despite numerous efforts to elucidate its physiological role, the exact biological function remains unknown. Many lines of evidences indicate that prion is a copper binding protein and thus involved in the copper metabolism. Prion protein is not expressed only in mammals but also in other species such as birds, reptiles and fishes. However, it is noteworthy to point out that prion diseases are only observed in mammals while they seem to be spared to other species. The chicken prion protein (chPrP C) shares about 30% of identity in its primary sequence with mammal PrP C. Both types of proteins have an N-terminal domain endowed with tandem amino acid repeats (PHNPGY in the avian protein, PHGGGWQ in mammals), followed by a highly conserved hydrophobic core. Furthermore, NMR studies have highlighted a similar globular domain containing three alpha-helices, one short 3(10)-helix and a short antiparallel beta-sheet. Despite this structural similarity, it should be noted that the normal isoform of mammalian PrP C is totally degraded by proteinase K, while avian PrP C is not, thereby producing N-terminal domain peptide fragments stable to further proteolysis. Notably, the hexarepeat domain is considered essential for protein endocytosis, and it is supposed to be the analogous copper-binding octarepeat region of mammalian prion proteins. The number of copper binding sites, the affinity and the coordination environment of metal ions are still matter of discussion for both mammal and avian proteins. In this review, we summarize the similarities and the differences between mammalian and avian prion proteins, as revealed by studies carried out on the entire protein and related peptide fragments, using a range of experimental and computational approaches. In addition, we report the metal-driven conformational alteration, copper binding modes and the superoxide dismutase-like (SOD-like) activity of the related copper(II) complexes.

Molecular characterization of chicken prion proteins by C-terminal-specific monoclonal antibodies

We obtained seven monoclonal antibodies (mAbs) against chicken cellular prion protein (ChPrP C) by immunizing BALB/c mice with recombinant prion protein (rChPrP). Of the seven mAbs, two mAbs (6 and 26) could recognize rChPrP, but not ChPrP C, in chicken brain lysate via Western blot (WB) analysis. Three C-terminal linear epitopes (AAANQTEVEM, RWWS and SPVPQD) were identified in ChPrP amino acids by pepspot analysis with five mAbs. The mAbs recognizing the C-terminal epitopes in ChPrP C predominantly reacted with the N-terminal truncated ChPrP C in WB analysis, which differed from the reaction with N-terminal proline/glycine-rich repeats recognizing rabbit polyclonal antibody. These mAbs will soon be available as a useful tool to characterize the biology of ChPrP C in birds.

Copper(II) complexes with an avian prion N-terminal region and their potential SOD-like activity

Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies were carried out on copper(II) complexes with chicken prion protein N-terminal fragments, Ac-(PHNPGY)(4)-NH(2), and the mutated residue, Ac-(PHNPGF)(4)-NH(2), to assess the role of tyrosine in the copper coordination. Both thermodynamic and spectroscopic results indicate that chicken prion fragments are not able to bind more than two copper ions and only with the involvement of side chain tyrosine groups. The prevailing complex shows one copper ion bound to four imidazole nitrogen atoms in the 1:1 metal to ligand ratio systems. The superoxide dismutase (SOD)-like activity of copper(II) complexes with the avian peptides and mammal analogue, Ac-(PHGGGWGQ)(4)-NH(2), was also investigated by means of Pulse radiolysis. The copper(II) complexes with avian peptides do not display SOD-like activity, while very low activity has been detected for the copper(II) complexes with mammalian tetraoctarepeat.

A comparative molecular dynamics study on thermostability of human and chicken prion proteins

To compare the thermostabilities of human and chicken normal cellular prion proteins (HuPrPC and CkPrPC), molecular dynamics (MD) simulations were performed for both proteins at an ensemble level (10 parallel simulations at 400K and 5 parallel simulations at 300K as a control). It is found that the thermostability of HuPrPC is comparable with that of CkPrPC, which implicates that the non-occurrence of prion diseases in non-mammals cannot be completely attributed to the thermodynamic properties of non-mammalian PrPC.

Structure and Stability of the CuII Complexes with Tandem Repeats of the Chicken Prion

Prion protein (PrP) misfolding is one of the pivotal issues in understanding the rudiments of neurodegenerative disorders. The conformational change of mammalian cellular PrP to scrapie PrP is caused by an unknown agent, but there is reasonable evidence supporting the key role of copper ions in this process. The structure of the avian PrP was found to be very similar to the mammalian protein, although there is only 30% homology in the secondary structure. This work shows that copper ions are very effectively bound by hexarepeat fragments of chicken prion protein, although not as effectively as it was found in the case of mammalian protein. By means of potentiometric and spectroscopic techniques (nuclear magnetic resonance, circular dichroism, UV-vis, and electronic paramagnetic resonance), it was shown that Cu(II) ions coordinate to the chicken PrP hexapeptide domain in physiological pH via imidazole nitrogen donors of His residue(s). The binding pattern changes the structure of peptide involved, indicating a possible impact of Cu(II) ions in the biology and pathology of nonmammalian PrP, which could be similar to that found for mammalian PrP. The present study shows that, similar to the human prion octapeptide repeats, chicken prion hexapeptide repeats might bind copper ions in two different ways, depending on the number of repeats and metal/ligand molar ratio: (i) an intra-repeat coordination mode in which copper ion is chelated by His imidazole and deprotonated amide nitrogen in monomeric peptide and (ii) an inter-repeat coordination mode in which a polymeric peptide ligand (dimer and trimer) forms polyimidazole complexes that are very stable at physiological pH. Two proline residues inserted into the hexapeptide unit have a critical impact on the metal-binding ability.