Biomedical subjects

Andrew C Gill

Publications and source records attributed to Andrew C Gill.

12 recordsLinked to original sources

PubMed2006

A novel, resistance-linked ovine PrP variant and its equivalent mouse variant modulate the in vitro cell-free conversion of rPrP to PrP(res).

Prion diseases are associated with the conversion of the normal cellular prion protein, PrP(c), to the abnormal, disease-associated form, PrP(Sc). This conversion can be mimicked in vitro by using a cell-free conversion assay. It has recently been shown that this assay can be modified to use bacterial recombinant PrP as substrate and mimic the in vivo transmission characteristics of rodent scrapie. Here, it is demonstrated that the assay replicates the ovine polymorphism barriers of scrapie transmission. In addition, the recently identified ovine PrP variant ARL(168)Q, which is associated with resistance of sheep to experimental BSE, modulates the cell-free conversion of ovine recombinant PrP to PrP(res) by three different types of PrP(Sc), reducing conversion efficiencies to levels similar to those of the ovine resistance-associated ARR variant. Also, the equivalent variant in mice (L(164)) is resistant to conversion by 87V scrapie. Together, these results suggest a significant role for this position and/or amino acid in conversion.

Amino Acid Substitution↗

PubMed2006

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

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) 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 PrP(C) and PrP(Sc) 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 PrP(C) to PrP(Sc), 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.

Animals↗

PubMed2005

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" PrP(Sc) 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 PrP(Sc) recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a beta-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 PrP(Sc), and the other to classical PrP(Sc). Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP(Sc) 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.

Animals↗

PubMed2005

Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate.

Signals delivered by the CD40 ligand, CD154, have crucial roles in immune responses in mammals, being required for development of germinal centres, maturation of T-dependent antibody responses, and generation of B-cell memory. To determine whether these functions were conserved in a non-mammalian species, a putative chicken CD 154 cDNA was used to make an oligomeric fusion protein, and to raise monoclonal antibodies. The antibodies detected surface expression on activated T-cells. The fusion protein detected expression of a receptor on B-cells, thrombocytes and macrophages. Biological effects of the fusion protein included induction of NO synthesis in a macrophage cell line, enhancement of splenic B-cell survival, and induction of apoptosis in a bursal lymphoma cell line. These observations demonstrated substantial functional equivalence with mammalian CD 154 and thus provided evidence for the early evolutionary emergence of the set of functions associated with this molecule, and its central role in the vertebrate immune system.

Amino Acid Sequence↗

PubMed2005

Calcium binding activity of the epidermal growth factor-like domains of the apicomplexan microneme protein EtMIC4.

Microneme proteins are secreted from apicomplexan parasites during invasion of host cells and they play crucial roles in parasite-host cell adhesion. EtMIC4 is a 240 kDa transmembrane protein from Eimeria tenella that contains 31 tandemly arranged epidermal growth factor (EGF), like repeats within its extracellular domain. The majority of these repeats have calcium binding (cb) consensus sequences. Little is known about cbEGFs in apicomplexan parasites but their presence in microneme proteins suggests that they may contribute to parasite-host interactions. To investigate the potential role of cbEGFs we have expressed and correctly refolded a cbEGF triplet from EtMIC4 (cbEGF7-9) and demonstrated that this triplet binds calcium. Circular dichroism spectroscopic analysis of cbEGF7-9 demonstrates that the molecule undergoes a gradual change in conformation with increasing levels of calcium. In the presence of calcium, the triplet becomes resistant to proteolytic degradation by a variety of proteases, a characteristic feature of cbEGF repeats from higher eukaryotic proteins, such as fibrillin, suggesting that calcium binding induces the formation of a rigid conformation. Moreover, mass spectrometric mapping of the cleavage sites that are protected by calcium shows that these sites are located both close to and distant from the calcium binding sites, indicating that protection is not due to steric hindrance by calcium ions, but rather due to the overall conformation adopted by the triplet in the presence of calcium. Thus, the tandemly-arranged cbEGF repeats within EtMIC4 provide a mechanism whereby, in the calcium-rich extracellular environment, the molecule could adopt a protease-resistant, rigid structure that could favour its interaction with host cell ligands.

Amino Acid Sequence↗

PubMed2004

Raman optical activity demonstrates poly(L-proline) II helix in the N-terminal region of the ovine prion protein: implications for function and misfunction.

The aqueous solution structure of the full-length recombinant ovine prion protein PrP(25-233), together with that of the N-terminal truncated version PrP(94-233), have been studied using vibrational Raman optical activity (ROA) and ultraviolet circular dichroism (UVCD). A sharp positive band at approximately 1315 cm(-1) characteristic of poly(L-proline) II (PPII) helix that is present in the ROA spectrum of the full-length protein is absent from that of the truncated protein, together with bands characteristic of beta-turns. Although it is not possible similarly to identify PPII helix in the full-length protein directly from its UVCD spectrum, subtraction of the UVCD spectrum of PrP(94-233) from that of PrP(25-233) yields a difference UVCD spectrum also characteristic of PPII structure and very similar to the UVCD spectrum of murine PrP(25-113). These results provide confirmation that a major conformational element in the N-terminal region is PPII helix, but in addition show that the PPII structure is interspersed with beta-turns and that little PPII structure is present in PrP(94-233). A principal component analysis of the ROA data indicates that the alpha-helix and beta-sheet content, located in the structured C-terminal domain, of the full-length and truncated proteins are similar. The flexibility imparted by the high PPII content of the N-terminal domain region may be an essential factor in the function and possibly also the misfunction of prion proteins.

Amino Acid Sequence↗

PubMed2004

Structural analysis of Salmonella enterica effector protein SopD.

Salmonella outer protein D (SopD) is a type III secreted virulence effector protein from Salmonella enterica. Full-length SopD and SopD lacking 16 amino acids at the N-terminus (SopDDeltaN) have been expressed as fusions with GST in Escherichia coli, purified with a typical yield of 20-30 mg per litre of cell culture and crystallized. Biophysical characterization has been carried out mainly on SopDDeltaN. Analytical size exclusion chromatography shows that SopDDeltaN is monomeric and probably globular in aqueous solution. The secondary structure composition, calculated from the CD spectrum, is mixed (38% alpha-helix and 26% beta-strand). Sequence analysis indicates that SopD contains a coiled coil motif, as found in numerous other type III secretion system-associated proteins. This suggests that SopD has the potential for one or more heterotypic protein-protein interactions. Limited trypsin digestion of SopDDeltaN, monitored by both one-dimensional proton NMR spectroscopy and SDS-PAGE, shows that the protein has a large, protease-resistant core domain of 286 amino acid residues. This single-domain architecture suggests that SopD lacks a cognate chaperone. In crystallization trials, SopDDeltaN produced better crystals than either full-length SopD or trypsin-digested SopDDeltaN. Diffraction to 3.0 A resolution has so far been obtained from crystals of SopDDeltaN.

Bacterial Proteins↗

PubMed2004

Methionine 129 variant of human prion protein oligomerizes more rapidly than the valine 129 variant: implications for disease susceptibility to Creutzfeldt-Jakob disease.

The human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies, but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the misfolding pathway that leads to the formation of beta-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP to that of the valine 129 variant. We provide evidence for differences in the folding behavior between the two variants at the early stages of oligomer formation. We show that Met(129) has a higher propensity to form beta-sheet-rich oligomers, whereas Val(129) has a higher tendency to fold into alpha-helical-rich monomers. An equimolar mixture of both variants displayed an intermidate folding behavior. We show that the oligomers of both variants are initially a mixture of alpha- and beta-rich conformers that evolve with time to an increasingly homogeneous beta-rich form. This maturation process, which involves no further change in proteinase K resistance, occurs more rapidly in the Met(129) form than the Val(129) form. Although the involvement of such beta-rich oligomers in prion pathogenesis is speculative, the misfolding behavior could, in part, explain the higher susceptibility of individuals that are methionine homozygote to both sporadic and variant Creutzfeldt-Jakob disease.

Alleles↗

PubMed2004

Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei.

BopE is a type III secreted protein from Burkholderia pseudomallei, the aetiological agent of melioidosis. Like its Salmonella homologues SopE and SopE2, BopE is a guanine nucleotide exchange factor for Rho GTPases. It is thought that, in order to be secreted by the type III system, proteins must be unfolded or only partially folded. As part of a study of B. pseudomallei virulence proteins, we have expressed, purified and characterized the catalytic domain of BopE (amino acids 78-261). Analytical ultracentrifugation experiments in conjunction with analytical size exclusion chromatography show that BopE(78-261) is monomeric in aqueous solution. CD spectroscopy indicates that the protein is predominantly alpha-helical, with predicted secondary structure composition of 59% alpha-helix and 7% beta-strand. NMR spectroscopy confirms that BopE(78-261) adopts a single, stable conformation. In differential scanning calorimetry experiments, thermal denaturation of BopE(78-261) (T(m) 52 degrees C) is reversible. Also, the secondary structure composition of BopE(78-261) changes little over a range of pH values from 3.5 to 10.5. BopE may therefore fold spontaneously to a functional form upon secretion into the host cell cytoplasm, and retains a native or native-like fold in varied environments. These properties are likely to be advantageous for a secreted bacterial effector protein.

Amino Acid Sequence↗

PubMed2003

A new perspective on beta-sheet structures using vibrational Raman optical activity: from poly(L-lysine) to the prion protein.

The vibrational Raman optical activity (ROA) spectrum of a polypeptide in a model beta-sheet conformation, that of poly(l-lysine), was measured for the first time, and the alpha-helix --> beta-sheet transition monitored as a function of temperature in H(2)O and D(2)O. Although no significant population of a disordered backbone state was detected at intermediate temperatures, some side chain bands not present in either the alpha-helix or beta-sheet state were observed. The observation of ROA bands in the extended amide III region assigned to beta-turns suggests that, under our experimental conditions, beta-sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-lysine) are different from those observed in typical beta-sheet proteins and may be characteristic of an extended flat multistranded beta-sheet, which is unlike the more irregular and twisted beta-sheet found in most proteins. However, a reduced isoform of the truncated ovine prion protein PrP(94-233) that is rich in beta-sheet shows amide I and amide II ROA bands similar to those of beta-sheet poly(L-lysine), which suggests that the C-terminal domain of the prion protein is able to support unusually flat beta-sheets. A principal component analysis (PCA) that identifies protein structural types from ROA band patterns provides a useful representation of the structural relationships among the polypeptide and protein states considered in the study.

Animals↗

PubMed2003

Stability and conformational properties of doppel, a prion-like protein, and its single-disulphide mutant.

Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the alpha-->beta transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95-148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced beta-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrP(C)-->PrP(Sc)). Furthermore, no direct interaction of either doppel protein with prion protein, either in the alpha-form or the beta-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the alpha-->beta transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.

Animals↗

PubMed2002

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, PrP(Sc). 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.

Carbohydrate Sequence↗