A quantitative characterization of interaction between prion protein with nucleic acids

Binding of recombinant prion protein with small highly structured RNAs, prokaryotic and eukaryotic prion protein mRNA pseudoknots, tRNA and polyA has been studied by the change in fluorescence anisotropy of the intrinsic tryptophan groups of the protein. The affinities of these RNAs to the prion protein and the number of sites where the protein binds to the nucleic acids do not vary appreciably although the RNAs have very different compositions and structures. The binding parameters do not depend upon pH of the solution and show a poor co-operativity. The reactants form larger nucleoprotein complexes at pH 5 compared to that at neutral pH. The electrostatic force between the protein and nucleic acids dominates the binding interaction at neutral pH. In contrast, nucleic acid interaction with the incipient nonpolar groups exposed from the structured region of the prion protein dominates the reaction at pH 5. Prion protein of a particular species forms larger complexes with prion protein mRNA pseudoknots of the same species. The structure of the pseudoknots and not their base sequences probably dominates their interaction with prion protein. Possibilities of the conversion of the prion protein to its infectious form in the cytoplasm by nucleic acids have been discussed.     

General features of the energetics of complex formation between ligand and nucleic acids

The analysis of the energy contributions of various physical factors to the complex formation between biologically active compounds and nucleic acids in aqueous solution was performed. A comparison of the energy parameters was made for ligand-ligand, intercalator-DNA, MGB-DNA and ligand-RNA groups. It was shown that the energetics of these reactions is of compensatory nature. Physical factors exerting the most pronounced influence on the energy parameters were identified. Correlation of the energy contributions to MGB-DNA complex formation and its biological effect was found.     

Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Prion protein interactions with nucleic acid: possible models for prion disease and prion function

Several models for the transmission and progression of prion diseases have arisen, evolving with the acquisition of new experimental results. It is generally accepted that the PrP^Sc protein is at least part of the infectious particle and the major protein component of the scrapie-associated fibrils (SAFs) that characterize the disease. An additional, unknown cofactor is most likely involved in transmission of the disease, perhaps by influencing the PrP^C PrP^Sc transition. This review relates experimental observations on the interactions of nucleic acids (NAs) and PrP with specific focus on alterations in structure. In particular, NAs appear to induce PrP^C to acquire some of the structural and biochemical characteristics of PrP^Sc. An updated hypothesis is related wherein NAs, on the basis of their structure, act in the PrP^C PrP^Sc transformation by serving as catalysts and/or chaperones and not by encoding genetic information.     

Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.     

Characterization of a complex formed between human plasminogen and recombinant sheep prion: pressure and thermal sensitivity of complex formation.

Scrapie is thought to be caused by one or more conformations of a proteinacious particle called a prion. The infectious form(s) is referred to as the scrapie form of the prion protein (PrPsc) whereas a benign form, the cellular conformer, is referred to as PrPC. The cellular conformation of the sheep prion protein formed a 1:1 complex with human plasminogen. The complex precipitated at 0 degrees C (Ksp = 17* 10(-12) M2). This precipitation reaction was sensitive to both temperature and pressure. When subjected to hydrostatic pressure the precipitate dissolved. At 25 degrees C the complex was soluble with a dissociation constant of about 10(-7) M as determined by isothermal titration calorimetry. Absorption, fluorescence and circular dichroism spectroscopy showed that neither protein, in the complex, underwent a detectable structural change so long as proteolytic inhibitors were present. In the absence of proteolytic inhibitors, plasminogen slowly cleaved the prion protein.     

Modeling a prion protein dimer: predictions for fibril formation

Models of structural transition in prion protein (PrP) focus on the domain visualised by solution NMR. Accumulating evidence suggests that the adjacent and highly conserved nonpolar segment, as well as PrP-membrane interactions, should also be considered. Calculations predict that membrane-induced structural destabilisation is mediated by stabilisation of the unfolded form. Comparative analysis of PrP structures leads to a model for PrP dimerisation that incorporates the nonpolar segment. A prediction that PrP will interact with the PrP-like protein (Dpl) to form a heterodimer, but that Dpl will not form a homodimer, can be tested. Modelling is discussed in the context of ataxias associated with the expression of Dpl or truncated PrP in transgenic animals lacking wild-type PrP. A PrP(C) dimer model forms the basis for considering the geometry of PrP(Sc) fibril formation.     

Bending and unwinding of nucleic acid by prion protein

Nucleic acid induces conformational changes in the prion protein (23-231 amino acids) to a structure resembling its pathological isoform. The prion protein, in turn, facilitates DNA strand transfer and acts as a DNA chaperone which is modulated by the N-terminal unstructured basic segment of the protein. Here we have studied the prion protein induced conformational changes in DNA using oligonucleotides covalently labeled with the energy donor fluorescein and the acceptor rhodamine moieties by fluorescence resonance energy transfer (FRET) and by thermal stability of the unlabeled oligonucleotides. The protein induces a strong FRET effect in the oligonucleotides evidenced from the simultaneous quenching of fluorescence intensity of the donor and increase in the fluorescence intensity of the acceptor, which indicate bending of the oligonucleotides by the prion protein. The energy transfer efficiency induced by the protein is greater for the larger oligonucleotide. The prion protein also induces significant structural destabilization of the oligonucleotides observed from the lowering of their melting temperatures in the presence of the protein. The truncated globular prion protein 121-231 fragment neither induces FRET effect on the oligonucleotides nor destabilizes their structures, indicating that the N-terminal segment of the prion protein is essential for the DNA bending process. Equilibrium binding and kinetics of FRET show that the protein binding to the oligonucleotides and their bending occur simultaneously. The DNA structural changes observed in the presence of the prion protein are similar to those caused by proteins involved in initiation and regulation for protein synthesis.     

Phosphoryl amino acids: Common origin for nucleic acids and protein

A series of compounds (DAP-AA) composed of an amino acid (AA) and a dialkyl phosphoryl group (DAP) is the basic elements of life chemistry. Self-catalysis of DAP-AA gives the self-assembly oligopeptides, even in aqueous medium at 38°C. The oligo-nucleotides could also be assembled from nucleosides' phosphorylation by DAP-AA. DAP-AA acts as the energy source as well as the phosphoryl donor for the synthesis of nuclic Acids and protein. A general expression for the self assembly system is proposed.     

Unique supramolecular assembly of a redox protein with nucleic acids onto hybrid bilayer: towards a dynamic DNA chip

Highly controlled supramolecular assemblies combining a genetically engineered redox protein, cytochrome b5, and modified oligonucleotides are presented. Modified b5 and DNA are covalently assembled through a hetero bifunctional cross-linker to give a unique hybrid molecular species. Moreover, the assembly includes a histidine tag head able to bind to modified phospholipids which lead to a new generation of self-assembled dynamic DNA chips. The interaction of the construction with a complementary oligonucleotide sequence can be monitored in real time by surface plasmon resonance using Biacore technology. The biochip, presented herein, features unique properties including tunable surface density of probes, very low non-specific interactions and optimization of hybridization efficiency. In addition, we demonstrated that the phase transition of the lipidic layer can modulate the dynamic of the association of the complex to the supported membrane. Potential applications of this new device are multiple including high sensitivity and high selectivity biochips, especially for studies of the DNA-ligands interactions in a biomimetic environment.     

Experimental approaches to the interaction of the prion protein with nucleic acids and glycosaminoglycans: Modulators of the pathogenic conversion

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrP(C)) into a misfolded, β-sheet-rich isoform (PrP(Sc)) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrP(Sc), with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrP(C) into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrP(C) and PrP(Sc) molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies.     

Interaction between DEAE-dextran and nucleic acids

DEAE-dextran at critical concentrations showed a precipitative action on nucleic acids. When the concentration of DEAE-dextran was increased above the critical level, the nucleic acids went back into solution and a colloidal state seemed to be attained. The properties of nucleic acids at the colloidal stage of the interaction with DEAE-dextran were investigated and it was found that:

• 1.1. RNA was no longer precipitable with alcohol;
• 2.2. the bases of the RNA retained their capacity to react with formaldehyde;
• 3.3. DNA was protected against deoxyribonuclease degradation;
• 4.4. RNA was only partially protected against ribonuclease degradation, and messenger RNA coated with DEAE-dextran retained to some extent its messenger function after ribonuclease degradation.

     

Interrelationships between polyamines and nucleic acids

Summary The distribution of radioactivity from ³H-putrescine was studied in intact and degenerated sciatic nerves, and spinal ganglia of rats by means of high resolution autoradiography. During the first three days after the administration of the labeled putrescine, the main proportion of radioactive material in the nerves was represented by spermidine and putrescine. Both, in intact and degenerating nerves, developed silver grains were deposited in all cellular components of the nervous tissue, the myelin sheath being markedly tagged. Perineural tissue was also labeled considerably, however, there was no significant amount of label in the extracellular space and in the collagen fibrils. The possible physiological significance of putrescine and spermidine in myelin and in other cellular components of nerves is discussed.Herrn Prof. Dr. W. Krücke zum 60. Geburtstag gewidmet.     

Hybridization of complementary and homologous peptide nucleic acid oligomers to a guanine quadruplex-forming RNA

Peptide nucleic acid (PNA) oligomers targeted to guanine quadruplex-forming RNAs can be designed in two different ways. First, complementary cytosine-rich PNAs can hybridize by the formation of Watson-Crick base pairs, resulting in hybrid PNA-RNA duplexes. Second, guanine-rich homologous PNAs can hybridize by the formation of G tetrads, resulting in hybrid PNA-RNA quadruplexes. UV thermal denaturation, circular dichroism, and fluorescence spectroscopy experiments were used to compare these two recognition modes and revealed 1:1 duplex formation for the complementary PNA and 2:1 (PNA2-RNA) quadruplex formation for the homologous PNA. Both hybrids were very stable, and hybridization was observed at low nanomolar concentrations. Hybrid quadruplex formation was equally efficient regardless of the PNA strand polarity, indicating a lack of interaction between the loop nucleobases on the PNA and RNA strands. The implications of this finding on sequence specificity as well as methods to improve affinity are also discussed.