Biomedical subjects

Ulrich Matthey

Publications and source records attributed to Ulrich Matthey.

7 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

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

Observing membrane protein diffusion at subnanometer resolution.

Single sodium-driven rotors from a bacterial ATP synthase were embedded into a lipid membrane and observed in buffer solution at subnanometer resolution using atomic force microscopy (AFM). Time-lapse AFM topographs show the movement of single proteins within the membrane. Subsequent analysis of their individual trajectories, in consideration of the environment surrounding the moving protein, allow principal modes of the protein motion to be distinguished. Within one trajectory, individual proteins can undergo movements assigned to free as well as to obstacled diffusion. The diffusion constants of these two modes of motion are considerably different. Without the structural information about the membrane environment restricting the moving proteins, it would not be possible to reveal insight into these mechanisms. The high-resolution AFM topographs suggest that, in future studies, such data revealed under various physiological conditions will provide novel insights into molecular mechanisms that drive membrane protein assembly and supply excellent boundary conditions to model protein-protein arrangements.

Diffusion↗

PubMed2003

Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases.

The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself.

Bacterial Proteins↗

PubMed2003

Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1.

We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

Adenosine Triphosphate↗

PubMed2002

Molecular architecture of the undecameric rotor of a bacterial Na+-ATP synthase.

The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.

Amino Acid Sequence↗

PubMed2002

NMR investigations of subunit c of the ATP synthase from Propionigenium modestum in chloroform/methanol/water (4 : 4 : 1).

The subunit c from the ATP synthase of Propionigenium modestum was studied by NMR in chloroform/methanol/water (4 : 4 : 1). In this solvent, subunit c consists of two helical segments, comprised of residues L5 to I26 and G29 to N82, respectively. On comparing the secondary structure of subunit c from P. modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the alpha helical structure within the conserved GXGXGXGX motif was shortened from five to two residues, the prominent interruption of the alpha helical structure in the cystoplasmic loop region was not apparent, and neither was there a break in the alpha helix after the sodium ion-binding Glu65 residue. The folding of subunit c of P. modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that subunit c of P. modestum did not adopt a stable hairpin structure like subunit c of E. coli.

Amino Acid Sequence↗