Andrew C. Gill

Characterization of 2′-Fluoro-RNA Aptamers That Bind Preferentially to Disease-associated Conformations of Prion Protein and Inhibit Conversion

We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2′-fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrPSc than for recombinant PrPC and inhibited the accumulation of PrPres in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from non-specific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, α-helix-rich and β-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the β-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the α-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23–110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the α-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is β-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.

Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob Disease.

In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89–230 (rPrP 89‐230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike “classical” PrPSc produced in vivo, the amyloid fibrils generated in vitro were more proteinase‐K sensitive. Here we demonstrate that the amyloid form contains a proteinase K‐resistant core composed only of residues 152/153–230 and 162–230. The PK‐resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrPSc recently identified in patients with sporadic Creutzfeldt‐Jakob disease (CJD). Remarkably, this core is sufficient for self‐propagating activity in vitro and preserves a β‐sheet‐rich fibrillar structure. Full‐length recombinant PrP 23‐230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrPSc, and the other to classical PrPSc. Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrPSc with a short PK‐resistant C‐terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.

Mass Spectroscopic Characterization of the Coronavirus Infectious Bronchitis Virus Nucleoprotein and Elucidation of the Role of Phosphorylation in RNA Binding by Using Surface Plasmon Resonance

ABSTRACT Phosphorylation of the coronavirus nucleoprotein (N protein) has been predicted to play a role in RNA binding. To investigate this hypothesis, we examined the kinetics of RNA binding between nonphosphorylated and phosphorylated infectious bronchitis virus N protein with nonviral and viral RNA by surface plasmon resonance (Biacore). Mass spectroscopic analysis of N protein identified phosphorylation sites that were proximal to RNA binding domains. Kinetic analysis, by surface plasmon resonance, indicated that nonphosphorylated N protein bound with the same affinity to viral RNA as phosphorylated N protein. However, phosphorylated N protein bound to viral RNA with a higher binding affinity than nonviral RNA, suggesting that phosphorylation of N protein determined the recognition of virus RNA. The data also indicated that a known N protein binding site (involved in transcriptional regulation) consisting of a conserved core sequence present near the 5′ end of the genome (in the leader sequence) functioned by promoting high association rates of N protein binding. Further analysis of the leader sequence indicated that the core element was not the only binding site for N protein and that other regions functioned to promote high-affinity binding.

Precursor ion scanning for detection and structural characterization of heterogeneous glycopeptide mixtures

The structure of N-linked glycans is determined by a complex, anabolic, intracellular pathway but the exact role of individual glycans is not always clear. Characterization of carbohydrates attached to glycoproteins is essential to aid understanding of this complex area of biology. Specific mass spectral detection of glycopeptides from protein digests may be achieved by on-line HPLC-MS, with selected ion monitoring (SIM) for diagnostic product ions generated by cone voltage fragmentation, or by precursor ion scanning for terminal saccharide product ions, which can yield the same information more rapidly. When glycosylation is heterogeneous, however, these approaches can result in spectra that are complex and poorly resolved. We have developed methodology, based around precursor ion scanning for ions of high m/z, that allows site specific detection and structural characterization of glycans at high sensitivity and resolution. These methods have been developed using the standard glycoprotein, fetuin, and subsequently applied to the analysis of the N-linked glycans attached to the scrapie-associated prion protein, PrPSc. These glycans are highly heterogeneous and over 30 structures have been identified and characterized site specifically. Product ion spectra have been obtained on many glycopeptides confirming structure assignments. The glycans are highly fucosylated and carry Lewis X or sialyl Lewis X epitopes and the structures are in-line with previous results. [Abbreviations: Hex—Hexose, C6H12O6 carbohydrates, including mannnose and galactose; HexNAc—N-acetylhexosamine, C8H15NO6 carbohydrates, including N-acetylglucosamine and N-acetylgalactosamine; GlcNAc—N-acetylglucosamine; GalNAc—N-acetylgalactosamine; Fuc—Fucose; NeuAC—N-acetylneuraminic acid or sialic acid; TSE—Transmissible Spongiform Encephalopathy.]

Post-translational hydroxylation at the N-terminus of the prion protein reveals presence of PPII structure in vivo.

The transmissible spongiform encephalopathies are characterized by conversion of a host protein, PrPC (cellular prion protein), to a protease‐resistant isoform, PrPSc (prion protein scrapie isoform). The importance of the highly flexible, N‐terminal region of PrP has recently become more widely appreciated, particularly the biological activities associated with its metal ion‐binding domain and its potential to form a poly(L‐proline) II (PPII) helix. Circular dichroism spectroscopy of an N‐terminal peptide, PrP37–53, showed that the PPII helix is formed in aqueous buffer; as it also contains an Xaa–Pro–Gly consensus sequence, it may act as a substrate for the collagen‐modifying enzyme prolyl 4‐hydroxylase. Direct evidence for this modification was obtained by mass spectrometry and Edman sequencing in recombinant mouse PrP secreted from stably transfected Chinese hamster ovary cells. Almost complete conversion of proline to 4‐hydroxyproline occurs specifically at residue Pro44 of this murine protein; the same hydroxylated residue was detected, at lower levels, in PrPSc from the brains of scrapie‐infected mice. Cation binding and/or post‐translational hydroxylation of this region of PrP may regulate its role in the physiology and pathobiology of the cell.

Conformations of biopolymers in the gas phase: a new mass spectrometric method

An in-the-canal hearing aid has a shell having an inner end to be positioned in the canal adjacent the users eardrum, and a faceplate located outwardly of the inner end but still adapted to be recessed within the ear canal in use. A protruding portion of the shell extends outwardly past the faceplate into the concha bowl and serves the dual purpose of both anchoring the hearing aid in the ear so that it cannot work its way down the ear canal, and providing a grip to facilitate insertion and removal of the hearing aid. The protruding portion is preferably cut back close to the faceplate at one side of the faceplate to facilitate battery insertion and removal, and may contain an aperture or a hook-like portion to facilitate gripping. A vent to vent the hearing aid may extend outwardly on the protruding portion to a position adjacent the rim of the protruding portion, to space the outer vent opening away from the microphone opening on the faceplate, to reduce the likelihood of feedback.

Synthesis and structural characterization of a mimetic membrane-anchored prion protein.

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrPSc) of the host encoded prion protein (PrPC) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrPC and PrPSc have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrPC to PrPSc, but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C‐terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP–GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane‐anchored PrP. We show that the structure of PrP–GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor‐free forms of PrP represent the structure of cellular, membrane‐anchored PrP. The availability of a lipid‐anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.

The presence of valine at residue 129 in human prion protein accelerates amyloid formation

The polymorphism at residue 129 of the human PRNP gene modulates disease susceptibility and the clinico‐pathological phenotypes in human transmissible spongiform encephalopathies. The molecular mechanisms by which the effect of this polymorphism are mediated remain unclear. It has been shown that the folding, dynamics and stability of the physiological, α‐helix‐rich form of recombinant PrP are not affected by codon 129 polymorphism. Consistent with this, we have recently shown that the kinetics of amyloid formation do not differ between protein containing methionine at codon 129 and valine at codon 129 when the reaction is initiated from the α‐monomeric PrPC‐like state. In contrast, we have shown that the misfolding pathway leading to the formation of β‐sheet‐rich, soluble oligomer was favoured by the presence of methionine, compared with valine, at position 129. In the present work, we examine the effect of this polymorphism on the kinetics of an alternative misfolding pathway, that of amyloid formation using partially folded PrP allelomorphs. We show that the valine 129 allelomorph forms amyloids with a considerably shorter lag phase than the methionine 129 allelomorph both under spontaneous conditions and when seeded with pre‐formed amyloid fibres. Taken together, our studies demonstrate that the effect of the codon 129 polymorphism depends on the specific misfolding pathway and on the initial conformation of the protein. The inverse propensities of the two allelomorphs to misfold in vitro through the alternative oligomeric and amyloidogenic pathways could explain some aspects of prion diseases linked to this polymorphism such as age at onset and disease incubation time.

Domains of invasion organelle proteins from apicomplexan parasites are homologous with the Apple domains of blood coagulation factor XI and plasma pre-kallikrein and are members of the PAN module superfamily.

Micronemes are specialised organelles, found in all apicomplexan parasites, which secrete molecules that are essential for parasite attachment to and invasion of host cells. Regions of several microneme proteins have sequence similarity to the Apple domains (A‐domains) of blood coagulation factor XI (FXI) and plasma pre‐kallikrein (PK). We have used mass spectrometry on a recombinant‐expressed, putative A‐domain from the microneme protein EtMIC5 from Eimeria tenella, to demonstrate that three intramolecular disulphide bridges are formed. These bridges are analogous to those that stabilise A‐domains in FXI and PK. The data confirm that the apicomplexan domains are structural homologues of A‐domains and are therefore novel members of the PAN module superfamily, which also includes the N‐terminal domains of members of the plasminogen/hepatocyte growth factor family. The role of A‐domains/PAN modules in apicomplexan parasites is not known, but their presence in the microneme suggests that they may be important for mediating protein–protein or protein–carbohydrate interactions during parasite attachment and host cell invasion.

Physiological Functions of the Cellular Prion Protein

The prion protein, PrPC, is a small, cell-surface glycoprotein notable primarily for its critical role in pathogenesis of the neurodegenerative disorders known as prion diseases. A hallmark of prion diseases is the conversion of PrPC into an abnormally folded isoform, which provides a template for further pathogenic conversion of PrPC, allowing disease to spread from cell to cell and, in some circumstances, to transfer to a new host. In addition to the putative neurotoxicity caused by the misfolded form(s), loss of normal PrPC function could be an integral part of the neurodegenerative processes and, consequently, significant research efforts have been directed toward determining the physiological functions of PrPC. In this review, we first summarise important aspects of the biochemistry of PrPC before moving on to address the current understanding of the various proposed functions of the protein, including details of the underlying molecular mechanisms potentially involved in these functions. Over years of study, PrPC has been associated with a wide array of different cellular processes and many interacting partners have been suggested. However, recent studies have cast doubt on the previously well-established links between PrPC and processes such as stress-protection, copper homeostasis and neuronal excitability. Instead, the functions best-supported by the current literature include regulation of myelin maintenance and of processes linked to cellular differentiation, including proliferation, adhesion, and control of cell morphology. Intriguing connections have also been made between PrPC and the modulation of circadian rhythm, glucose homeostasis, immune function and cellular iron uptake, all of which warrant further investigation.