Biomedical subjects

Ralph Zahn

Publications and source records attributed to Ralph Zahn.

11 recordsLinked to original sources

PubMed2006

Prion protein in milk.

BACKGROUND: Prions are known to cause transmissible spongiform encephalopathies (TSE) after accumulation in the central nervous system. There is increasing evidence that prions are also present in body fluids and that prion infection by blood transmission is possible. The low concentration of the proteinaceous agent in body fluids and its long incubation time complicate epidemiologic analysis and estimation of spreading and thus the risk of human infection. This situation is particularly unsatisfactory for food and pharmaceutical industries, given the lack of sensitive tools for monitoring the infectious agent. METHODOLOGY/PRINCIPAL FINDINGS: We have developed an adsorption matrix, Alicon PrioTrap, which binds with high affinity and specificity to prion proteins. Thus we were able to identify prion protein (PrP(C))--the precursor of prions (PrP(Sc))--in milk from humans, cows, sheep, and goats. The absolute amount of PrP(C) differs between the species (from microg/l range in sheep to ng/l range in human milk). PrP(C) is also found in homogenised and pasteurised off-the-shelf milk, and even ultrahigh temperature treatment only partially diminishes endogenous PrP(C) concentration. CONCLUSIONS/SIGNIFICANCE: In view of a recent study showing evidence of prion replication occurring in the mammary gland of scrapie infected sheep suffering from mastitis, the appearance of PrP(C) in milk implies the possibility that milk of TSE-infected animals serves as source for PrP(Sc).

Animals↗

PubMed2006

Observation of intermediate states of the human prion protein by high pressure NMR spectroscopy.

BACKGROUND: Prions as causative agents of transmissible spongiform encephalopathies (TSEs) in humans and animals are composed of the infectious isomer, PrPSc, of the cellular prion protein, PrPC. The conversion and thus the propensity of PrPC to adopt alternative folds leads to the species-specific propagation of the disease. High pressure is a powerful tool to study the physico-chemical properties of proteins as well as the dynamics and structure of folding intermediates. RESULTS: Conformational intermediates of the human prion protein huPrPC were characterized by a combination of hydrostatic pressure (up to 200 MPa) with two-dimensional NMR spectroscopy. All pressure effects showed to be reversible and there is virtually no difference in the overall pressure response between the folded core of the N-terminal truncated huPrPC(121-230) and the full-length huPrPC(23-230). The only significant differences in the pressure response of full-length and truncated PrP suggest that E168, H187, T192, E207, E211 and Y226 are involved in a transient interaction with the unfolded N-terminus. High-pressure NMR spectroscopy indicates that the folded core of the human prion protein occurs in two structural states N1 and N2 in solution associated with rather small differences in free enthalpies (3.0 kJ/mol). At atmospheric pressure approximately 29% of the protein are already in the pressure favored conformation N2. There is a second process representing two possible folding intermediates I1 and I2 with corresponding average free enthalpies of 10.8 and 18.6 kJ/mol. They could represent preaggregation states of the protein that coexist at ambient pressure with a very small population of approximately 1.2% and less than 0.1%. Further the pressure response of the N-terminus indicates that four different regions are in a fast equilibrium with non-random structural states whose populations are shifted by pressure. CONCLUSION: We identified pressure stabilized folding intermediates of the human prion protein. The regions reflecting most strongly the transition to the intermediate states are the beta1/alpha1-loop and the solvent exposed side of alpha3. The most pressure-sensitive region (representing mainly intermediate I1) is the loop between beta-strand 1 and alpha-helix 1 (residue 139-141), indicating that this region might be the first entry point for the infectious conformer to convert the cellular protein.

Humans↗

PubMed2006

Enhanced stability of human prion proteins with two disulfide bridges.

We compare the folding equilibrium of the globular domain of the human prion protein with two variants of this domain, for which an additional disulfide bond was introduced into the location where it is found in the naturally occurring doppel protein. We find that the unfolding transition midpoint of the variants is shifted toward higher denaturant concentration, indicating that the engineered disulfide bond significantly stabilizes the global protein structure. Our results further reveal that the two-disulfide variant proteins, while possessing the same global fold as the wild-type, display marked differences in their folding pathway-in particular, the absence of a characteristic alpha-helix to beta-sheet transition, which is a fundamental feature associated with misfolding of proteins into amyloid fibrils, especially in the context of prion diseases. These surprising characteristics of disulfide mutant prion proteins have important implications for the understanding of the generic aberrant processes leading to amyloid fibril formation and protein aggregation, as well as providing insight into possible therapeutic strategies.

Binding Sites↗

PubMed2006

Gender differences in acute non-ST-segment elevation myocardial infarction.

To assess gender-based differences in presentation and outcome after non-ST-elevation myocardial infarction (NSTEMI) in clinical practice, this study examined data from the Acute Coronary Syndrome registry, which enrolled 16,817 patients from 2000 through the end of 2002, 6,358 of them with NSTEMIs (34.1% women). Women with NSTEMIs were 7.5 years older, had a history of myocardial infarction and percutaneous coronary intervention or coronary artery bypass graft less often, and were less likely to have smoked. They more often had a history of systemic hypertension and diabetes mellitus, but this difference was due to their older age. Reperfusion therapy was performed less often in women, which still was significant after adjustment for baseline variables (odds ratio 0.71, 95% confidence interval 0.63 to 0.80). Clopidogrel was given less often in women (43.4% vs 56%). After adjustment for age, gender differences in medical therapy with statins, aspirin, and beta blockers were not significant. Hospital mortality was 1.7 times greater in women. This difference was not significant after adjustment for age (odds ratio 1.07, 95% confidence interval 0.84 to 1.35). Women had greater crude long-term mortality, but after age adjustment, this difference was no longer significant (odds ratio 0.92, 95% confidence interval 0.76 to 1.11). In conclusion, women with NSTEMIs were older than men and thus more often had concomitant diseases but less often had a history of myocardial infarction or coronary artery bypass grafts. They less often received acute percutaneous coronary intervention and less often were treated with clopidogrel. However, there was no difference in age-adjusted mortality in women.

Aged↗

PubMed2006

Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions.

A soluble, oligomeric beta-sheet-rich conformational variant of recombinant full-length prion protein, PrP beta, was generated that aggregates into amyloid fibrils, PrP betaf. These fibrils have physico-chemical and structural properties closely similar to those of pathogenic PrP Sc in scrapie-associated fibrils and prion rods, including a closely similar proteinase K digestion pattern and Congo red birefringence. The conformational transition from PrP C to PrP beta occurs at pH 5.0 in bicellar solutions containing equimolar mixtures of dihexanoyl-phosphocholine and dimyristoyl-phospholipids, and a small percentage of negatively charged dimyristoyl-phosphoserine. The same protocol was applicable to human, cow, elk, pig, dog and mouse PrP. Comparison of full-length hPrP 23-230 with the N-terminally truncated human PrP fragments hPrP 90-230, hPrP 96-230, hPrP 105-230 and hPrP 121-230 showed that the flexible peptide segment 105-120 must be present for the generation of PrP beta. Dimerization of PrP C represents the rate-limiting step of the PrP C-to-PrP beta conformational transition, which is dependent on the amino acid sequence. The activation enthalpy of dimerization is about 130 kJ/mol for the recombinant full-length human and bovine prion proteins, and between 260 and 320 kJ/mol for the other species investigated. The in vitro conversion assay described here permits direct molecular characterization of processes that might be closely related to conformational transitions of the prion protein in transmissible spongiform encephalopathies.

Amyloid↗

PubMed2006

Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein.

This study describes the conversion of murine PrP(C) by PrP(Sc) from three different mouse scrapie strains (ME7, 87V and 22A) and from a mouse-passaged bovine spongiform encephalopathy (BSE) strain (BSE/Bl6). This was demonstrated by a modified, non-radioactive, cell-free conversion assay using bacterial prion protein, which was converted into a proteinase K (PK)-resistant fragment designated PrP(res). Using this assay, newly formed PrP(res) could be detected by an antibody that discriminated de novo PrP(res) and the original PrP(Sc) seed. The results suggested that PrP(res) formation occurs in three phases: the first 48 h when PrP(res) formation is delayed, followed by a period of substantially accelerated PrP(res) formation and a plateau phase when a maximum concentration of PrP(res) is reached after 72 h. The conversion of prokaryotically expressed PrP(C) by ME7 and BSE prions led to unglycosylated, PK-digested, abnormal PrP(res) fragments, which differed in molecular mass by 1 kDa. Therefore, prion strain phenotypes were retained in the cell-free conversion, even when recombinant PrP(C) was used as the substrate. Moreover, co-incubation of ME7 and BSE prions resulted in equal amounts of both ME7- and BSE-derived PrP(res) fragments (as distinguished by their different molecular sizes) and also in a significantly increased total amount of de novo-generated PrP(res). This was found to be more than twice the amount of either strain when incubated separately. This result indicates a synergistic effect of both strains during cell-free conversion. It is not yet known whether such a cooperative action between BSE and scrapie prions also occurs in vivo.

Animals↗

PubMed2003

The octapeptide repeats in mammalian prion protein constitute a pH-dependent folding and aggregation site.

Structural studies of mammalian prion protein at pH values between 4.5 and 5.5 established that the N-terminal 100 residue domain is flexibly disordered. Here, we show that at pH values between 6.5 and 7.8, i.e. the pH at the cell membrane, the octapeptide repeats in recombinant human prion protein hPrP(23-230) encompassing the highly conserved amino acid sequence PHGGGWGQ are structured. The nuclear magnetic resonance solution structure of the octapeptide repeats at pH 6.2 reveals a new structural motif that causes a reversible pH-dependent PrP oligomerization. Within the aggregation motif the segments HGGGW and GWGQ adopt a loop conformation and a beta-turn-like structure, respectively. Comparison with the crystal structure of HGGGW-Cu(2+) indicates that the binding of copper ions induces a conformational transition that presumably modulates PrP aggregation. The knowledge that the cellular prion protein is immobilized on the cell surface along with our results suggests a functional role of aggregation in endocytosis or homophilic cell adhesion.

Binding Sites↗

PubMed2003

Antimalarial drug quinacrine binds to C-terminal helix of cellular prion protein.

Using NMR spectroscopy we show that the cellular prion protein constitutes a target for binding of various acridine and phenothiazine derivatives. We unambiguously map the quinacrine binding site of recombinant human prion protein to residues Tyr225, Tyr226, and Gln227 of helix alpha3, which is located near the "protein X" epitope. The millimolar dissociation constant of the complex suggests that in vivo inhibition of prion propagation occurs after 10000-fold concentration of quinacrine within endolysosomes.

Antimalarials↗

PubMed2003

Influence of pH on NMR structure and stability of the human prion protein globular domain.

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three alpha-helices, comprising residues 144-156, 174-194, and 200-228, and a short anti-parallel beta-sheet, comprising residues 128-131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix alpha1 is elongated at the C-terminal end with residues 153-156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix alpha 2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154-157 at the C-terminal end of helix alpha 1 and residues 161-164 of beta-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopathies.

Amino Acid Sequence↗

PubMed2003

NMR structure of a variant human prion protein with two disulfide bridges.

The nuclear magnetic resonance structure of the globular domain with residues 121-230 of a variant human prion protein with two disulfide bonds, hPrP(M166C/E221C), shows the same global fold as wild-type hPrP(121-230). It contains three alpha-helices of residues 144-154, 173-194 and 200-228, an anti-parallel beta-sheet of residues 128-131 and 161-164, and the disulfides Cys166-Cys221 and Cys179-Cys214. The engineered extra disulfide bond in the presumed "protein X"-binding site is accommodated with slight, strictly localized conformational changes. High compatibility of hPrP with insertion of a second disulfide bridge in the protein X epitope was further substantiated by model calculations with additional variant structures. The ease with which the hPrP structure can accommodate a variety of locations for a second disulfide bond within the presumed protein X-binding epitope suggests a functional role for the extensive perturbation by a natural second disulfide bond of the corresponding region in the human doppel protein.

Amino Acid Sequence↗

PubMedDate not supplied

A structurally deviant member of the Euplotes raikovi pheromone family: Er-23.

Pheromones of Euplotes raikovi form a homologous family of proteins with 37- to 40-amino acid residues, including six cysteines that form three strictly conserved disulfide bridges. The determination of the primary structure of the pheromone Er-23, which was isolated from cells derived from natural populations of E. raikovi that secrete the other known pheromones, has now revealed a novel structure type. The polypeptide chain of this pheromone contains 51 residues, 10 of which are cysteines presumably involved in the formation of five disulfide bridges, and lacks a carboxyl-terminal tail following the last cysteine of the sequence. The elongation of the Er-23 molecule is presumed to result from multiple events of gene duplication starting from an ancestral motif Xxx(2-4)-Cys-Xxx(5-7)-Cys.

Amino Acid Sequence↗