A.R. Clarke’s research while affiliated with University of Bristol and other places

Supplementary Figures S1-S7 and Supplementary Table S1.

Prion diseases are fatal neurodegenerative disorders with unique transmissible properties. The infectious and pathological agent is thought to be a misfolded conformer of the prion protein. Little is known about the initial events in prion infection because the infecting prion source has been immunologically indistinguishable from normal cellular prion protein (PrP(C)). Here we develop a unique cell system in which epitope-tagged PrP(C) is expressed in a PrP knockdown (KD) neuroblastoma cell line. The tagged PrP(C), when expressed in our PrP-KD cells, supports prion replication with the production of bona fide epitope-tagged infectious misfolded PrP (PrP(Sc)). Using this epitope-tagged PrP(Sc), we study the earliest events in cellular prion infection and PrP misfolding. We show that prion infection of cells is extremely rapid occurring within 1 min of prion exposure, and we demonstrate that the plasma membrane is the primary site of prion conversion.

In prion diseases, the misfolded protein aggregates are derived from cellular prion protein (PrP(C)). Numerous ligands have been reported to bind to human PrP(C) (huPrP), but none to the structured region with the affinity required for a pharmacological chaperone. Using equilibrium dialysis, we screened molecules previously suggested to interact with PrP to discriminate between those which did not interact with PrP, behaved as nonspecific polyionic aggregates or formed a genuine interaction. Those that bind could potentially act as pharmacological chaperones. Here we report that a cationic tetrapyrrole [Fe(III)-TMPyP], which displays potent antiprion activity, binds to the structured region of huPrP. Using a battery of biophysical techniques, we demonstrate that Fe(III)-TMPyP forms a 11 complex via the structured C terminus of huPrP with a K(d) of 4.5 +/- 2 muM, which is in the range of its IC(50) for curing prion-infected cells of 1.6 +/- 0.4 muM and the concentration required to inhibit protein-misfo

Prion propagation involves a conformational transition of the cellular form of prion protein (PrPC) to a disease-specific isomer (PrPSc), shifting from a predominantly alpha-helical conformation to one dominated by beta-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrPSc, is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant beta-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrPC conformation, whereas helix II exhibits little

Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (approximately 3,000 s(-1)), with the extrapolated folding rate constant at approximately 20 degrees C in physiological conditions reaching 20,000 s(-1). By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant phi-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP(C) conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state

Prion infection is characterized by the conversion of host cellular prion protein (PrP(C)) into disease-related conformers (PrP(Sc)) and can be arrested in vivo by passive immunization with anti-PrP monoclonal antibodies. Here, we show that the ability of an antibody to cure prion-infected cells correlates with its binding affinity for PrP(C) rather than PrP(Sc). We have visualized this interaction at the molecular level by determining the crystal structure of human PrP bound to the Fab fragment of monoclonal antibody ICSM 18, which has the highest affinity for PrP(C) and the highest therapeutic potency in vitro and in vivo. In this crystal structure, human PrP is observed in its native PrP(C) conformation. Interactions between neighboring PrP molecules in the crystal structure are mediated by close homotypic contacts between residues at position 129 that lead to the formation of a 4-strand intermolecular beta-sheet. The importance of this residue in mediating protein-protein contact could explain the genetic susceptibility and prion strain selection determined by polymorphic residue 129 in human prion disease, one of the strongest common susceptibility polymorphisms known in any human disease.

Enzymes have become important tools in several industries due to their ability to produce chirally pure and complex molecules with interesting biological properties. The NAD(+)-dependent LDH (lactate dehydrogenase) [bsLDH [Geobacillus stearothermophilus (formerly Bacillus stearothermophilus) LDH] from G. stearothermophilus and the NAD(+)-dependent FDH (formate dehydrogenase) [cmFDH (Candida methylica FDH)] enzyme from C. methylica are particularly crucial enzymes in the pharmaceutical industry and are related to each other in terms of NADH use and regeneration. LDH catalyses the interconversion of pyruvate (oxo acid) and lactate (alpha-hydroxy acid) using the NADH/NAD(+) pair as a redox cofactor. Employing LDH to reduce other oxo acids can generate chirally pure alpha-hydroxy acids of use in the production of pharmaceuticals. One important use of FDH is to regenerate the relatively expensive NADH cofactor that is used by NAD(+)-dependent oxidoreductases such as LDH. Both LDH and FDH from organisms of interest were previously cloned and overproduced. Therefore they are available at a low cost. However, both of these enzymes show disadvantages in the large-scale production of chirally pure compounds. We have applied two routes of protein engineering studies to improve the properties of these two enzymes, namely DNA shuffling and site-directed mutagenesis. Altering the substrate specificity of bsLDH by DNA shuffling and changing the coenzyme specificity of cmFDH by site-directed mutagenesis are the most successful examples of our studies. The present paper will also include the details of these examples together with some other applications of protein engineering regarding these enzymes.

Prion diseases are associated with accumulation of strain-dependent biochemically distinct, disease-related isoforms (PrP(Sc)) of host-encoded prion protein (PrP(C)). PrP(Sc) is characterised by increased beta-sheet content, detergent insolubility and protease resistance. Recombinant alpha-PrP adopts a PrP(C)-like conformation, while beta-PrP conformationally resembles PrP(Sc), to these we raised 81 monoclonal antibodies in Prnp(0/0) mice. The N-terminal residues 91-110 are highly immunogenic in beta-PrP-immunised mice and of (17/41) anti-beta-PrP antibodies that could be epitope-mapped, approximately 70%, recognised this segment. In contrast, only 3/40 anti-alpha-PrP antibodies could be mapped and none interacted with this region, instead recognising residues 131-150, 141-160 and 171-190. Native PrP(C) was recognised by both antibody groups, but only anti-beta-PrP antibodies directed to 91-110 residues recognised native PrP(Sc) with high affinity, where in addition, species heterogeneity was also evid

Prions, transmissible agents that cause Creutzfeldt-Jakob disease (CJD) and other prion diseases, are known to resist conventional sterilization procedures. Iatrogenic transmission of classical CJD via neurosurgical instruments is well documented and the involvement of lymphoreticular tissues in variant CJD (vCJD), together with the unknown population prevalence of asymptomatic vCJD infection, has led to concerns about transmission from a wide range of surgical procedures. To address this problem, conditions were sought that destroy PrP(Sc) from vCJD-infected human tissue and eradicate RML prion infectivity adsorbed onto surgical steel. Seven proteolytic enzymes were evaluated individually and in pairs at a range of temperatures and pH values and the additional effects of detergents, lipases and metal ions were assessed. A combination of proteinase K and Pronase, in conjunction with SDS, was shown to degrade PrP(Sc) material from highly concentrated vCJD-infected brain preparations to a level below det