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Biomedical subjects

Andrew Thompson

Publications and source records attributed to Andrew Thompson.

8 recordsLinked to original sources
PubMed2003

Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS.

A novel MS/MS-based analysis strategy using isotopomer labels, referred to as "tandem mass tags" (TMTs), for the accurate quantification of peptides and proteins is described. The new tags are designed to ensure that identical peptides labeled with different TMTs exactly comigrate in all separations. The tags require novel methods of quantification analysis using tandem mass spectrometry. The new tags and analysis methods allow peptides from different samples to be identified by their relative abundance with greater ease and accuracy than other methods. The new TMTs permit simultaneous determination of both the identity and relative abundances of peptide pairs using a collision induced dissociation (CID)-based analysis method. Relative abundance measurements made in the MS/MS mode using the new tags are accurate and sensitive. Compared to MS-mode measurements, a very high signal-to-noise ratio is achieved with MS/MS based detection. The new tags should be applicable to a wide variety of peptide isolation methods.

Isotopes↗
PubMed2003

Diverse fibrillar peptides directly bind the Alzheimer's amyloid precursor protein and amyloid precursor-like protein 2 resulting in cellular accumulation.

The Alzheimer's disease Abeta peptide can increase the levels of cell-associated amyloid precursor protein (APP) in vitro. To determine the specificity of this response for Abeta and whether it is related to cytotoxicity, we tested a diverse range of fibrillar peptides including amyloid-beta (Abeta), the fibrillar prion peptides PrP106-126 and PrP178-193 and human islet-cell amylin. All these peptides increased the levels of APP and amyloid precursor-like protein 2 (APLP2) in primary cultures of astrocytes and neurons. Specificity was shown by a lack of change to amyloid precursor-like protein 1, tau-1 and cellular prion protein (PrP(c)) levels. APP and APLP2 levels were elevated only in cultures exposed to fibrillar peptides as assessed by electron microscopy and not in cultures treated with non-fibrillogenic peptide variants or aggregated lipoprotein. We found that PrP106-126 and the non-toxic but fibril-forming PrP178-193 increased APP levels in cultures derived from both wild-type and PrP(c)-deficient mice indicating that fibrillar peptides up-regulate APP through a non-cytotoxic mechanism and irrespective of parental protein expression. Fibrillar PrP106-126 and Abeta peptides bound recombinant APP and APLP2 suggesting the accumulation of these proteins was mediated by direct binding to the fibrillated peptide. This was supported by decreased APP accumulation following extensive washing of the cultures to remove fibrillar aggregates. Pre-incubation of fibrillar peptide with recombinant APP18-146, the putative fibril binding site, also abrogated the accumulation of APP. These findings show that diverse fibrillogenic peptides can induce accumulation of APP and APLP2 and this mechanism could contribute to pathogenesis in neurodegenerative disorders.

Alzheimer Disease↗
PubMed2002

Alzheimer's disease amyloid beta and prion protein amyloidogenic peptides promote macrophage survival, DNA synthesis and enhanced proliferative response to CSF-1 (M-CSF).

Microglial cells, macrophage-lineage cells in the brain, are increased in amyloid-containing plaques in Alzheimer's disease (AD) and in the lesions of prion diseases. Recent studies suggest that microglia have a central role in turnover of amyloid in these diseases. We report here that synthetic amyloid beta (Abeta) 1-42 and prion protein (PrP) 106-126 peptides promote macrophage survival; they also induce macrophage DNA synthesis, particularly in the presence of sub-optimal concentrations of the growth factor, macrophage-colony stimulating factor (M-CSF or CSF-1). These responses are proposed to provide a means to increase brain microglia/macrophage numbers thereby enhancing subsequent inflammatory/immune responses. These fibrillogenic peptides join the list of aggregates having these effects on macrophages, indicating the generality of this type of response.

Amyloid beta-Peptides↗
PubMed2002

Structure of the S pilus periplasmic chaperone SfaE at 2.2 A resolution.

S pili are sialic acid binding hair-like appendages expressed by pathogenic strains of Escherichia coli. The presence of S pili has been implicated as a virulence factor in both urinary-tract infections and new-born meningitis. Assembly of S pili proceeds via the ubiquitous chaperone/usher pathway. Previously, structures of the homologous chaperones PapD and FimC involved in assembly of P and type-1 pili, respectively, have been solved. Here, the 2.2 A X-ray structure of the S pilus chaperone SfaE is reported. SfaE has the same overall L-shaped structure as PapD and FimC, with two immunoglobulin-like domains oriented at about a 90 degrees angle to each other. Conserved residues in the subunit-binding cleft known to be critical for chaperone function occupy essentially identical positions in SfaE, FimC and PapD. As in free PapD and FimC, the long F1-G1 loop connecting the two last strands of the N-terminal domain is disordered. SfaE crystallizes as a dimer with an extensive dimer interface involving the subunit-binding surfaces of the chaperone. Dimerization via these regions has previously been observed for PapD and might be a general side effect arising from the subunit-binding properties of periplasmic chaperones. The domain interface contains an extended hydrogen-bond network involving three invariant charged residues and two structurally conserved water molecules. It is suggested that disruption of the domain interactions may destabilize the N-terminal domain through exposure of three conserved hydrophobic residues, thereby promoting release of pilus subunits during pilus assembly.

Crystallization↗
PubMed2002

3-Carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa: MAD phasing with 80 selenomethionines.

The structure of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa was determined at 3.0 A resolution. Phase information was derived from a multiwavelength anomalous dispersion (MAD) experiment conducted at three wavelengths using crystals of fully substituted selenomethionine protein. However, the structure determination was not routine owing to the relatively poor quality of the diffraction data and the large number of twofolds in the unit cell. Eventually, 80 selenium sites were identified by the combined use of direct methods and real-space map interpretation. This represents one of the largest selenium substructures solved and used for phasing. Some of the difficulties in the structure determination and the methods used to address them are discussed.

Crystallography, X-Ray↗
PubMed2002

Crystal structure of an antiparallel DNA fragment with Hoogsteen base pairing.

We report here an alternative double-helical structure of the DNA molecule. It has been found in the d(ATA(Br)UAT) and d(ATATAT) sequences by single-crystal x-ray crystallography. This sequence is found not only in TATA boxes, but also in other regulatory regions of DNA. Bases of the two antiparallel strands form Hoogsteen pairs, with adenines in the syn conformation. The structure is related neither to those found in triple helices nor to parallel DNA duplexes. Its conformational parameters are very similar to those of duplex DNA in the B form. Both forms may coexist under physiological conditions, although the Hoogsteen pairing greatly influences the recognition sites on DNA. Our results demonstrate that an alternative to the classical B-DNA double helix is possible.

Base Pairing↗
PubMed2002

The structure of Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme, a beta propeller cycloisomerase.

Muconate lactonizing enzymes (MLEs) convert cis,cis-muconates to muconolactones in microbes as part of the beta-ketoadipate pathway; some also dehalogenate muconate derivatives of xenobiotic haloaromatics. There are three different MLE classes unrelated by evolution. We present the X-ray structure of a eukaryotic MLE, Neurospora crassa 3-carboxy-cis,cis-muconate lactonizing enzyme (NcCMLE) at 2.5 A resolution, with a seven-bladed beta propeller fold. It is related neither to bacterial MLEs nor to other beta propeller enzymes, but is structurally similar to the G protein beta subunit. It reveals a novel metal-independent cycloisomerase motif unlike the bacterial metal cofactor MLEs. Together, the bacterial MLEs and NcCMLE structures comprise a striking structural example of functional convergence in enzymes for 1,2-addition-elimination of carboxylic acids. NcCMLE and bacterial MLEs may enhance the reaction rate differently: the former by electrophilic catalysis and the latter by electrostatic stabilization of the enolate.

Amino Acid Sequence↗