Biomedical subjects

Kevin Pearson

Publications and source records attributed to Kevin Pearson.

7 recordsLinked to original sources

PubMed2005

The biotin-capture lipid affinity assay: a rapid method for determining lipid binding parameters for apolipoproteins.

The lipid affinity of plasma apolipoproteins is an important modulator of lipoprotein metabolism. Mutagenesis techniques have been widely used to modulate apolipoprotein lipid affinity for studying biological function, but the approach requires rapid and reliable lipid affinity assays to compare the mutants. Here, we describe a novel method that measures apolipoprotein binding to a standardized preparation of small unilamellar vesicles (SUVs) containing trace biotinylated and fluorescent phospholipids. After a 30 min incubation at various apolipoprotein concentrations, vesicle-bound protein is rapidly separated from free protein on columns of immobilized streptavidin in a 96-well microplate format. Vesicle-bound protein and lipid are eluted and measured in a fluorescence microplate reader for calculation of a dissociation constant and the maximum number of potential binding sites on the SUVs. Using human apolipoprotein A-I (apoA-I), apoA-IV, and mutants of each, we show that the assay generates binding constants that are comparable to other methods and is reproducible across time and apolipoprotein preparations. The assay is easy to perform and can measure triplicate binding parameters for up to 10 separate apolipoproteins in 3.5 h, consuming only 120 microg of apolipoprotein in total. The benefits and potential drawbacks of the assay are discussed.

Algorithms↗

PubMed2005

Specific sequences in the N and C termini of apolipoprotein A-IV modulate its conformation and lipid association.

Apolipoprotein (apoA-IV) is a 376-residue exchangeable apolipoprotein that may play a number of important roles in lipid metabolism, including chylomicron assembly, reverse cholesterol transport, and appetite regulation. In vivo, apoA-IV exists in both lipid-poor and lipid-associated forms, and the balance between these states may determine its function. We examined the structural elements that modulate apoA-IV lipid binding by producing a series of deletion mutants and determining their ability to interact with phospholipid liposomes. We found that the deletion of residues 333-343 strongly increased the lipid association rate versus native apoA-IV. Additional mutagenesis revealed that two phenylalanine residues at positions 334 and 335 mediated this lipid binding inhibitory effect. We also observed that residues 11-20 in the N terminus were required for the enhanced lipid affinity induced by deletion of the C-terminal sequence. We propose a structural model in which these sequences can modulate the conformation and lipid affinity of apoA-IV.

Amino Acid Sequence↗

PubMed2005

Bacterial expression and characterization of rat apolipoprotein E.

Apolipoprotein (apo) E is a protein involved in both lipid metabolism and neuroprotection. Recently, it has been suggested that apoE may play a role in the regulation of food intake and body weight in rodents. However, rodent plasma apoE is difficult to purify in reasonable amounts due to numerous time-consuming steps. To circumvent this, we created a bacterial expression system for the efficient production of large amounts of rat apoE. We inserted rat apoE DNA into the pET30 expression vector and overexpressed the proteins in Escherichia coli strain BL21 (DE3). A histidine tag present at the N-terminus allowed for easy purification of the recombinant protein. The tag was removed with an IgA protease (Igase) from Neisseria gonorrhoeae leaving the mature form of the protein. The use of Igase was important as several more common proteases routinely cleave apolipoproteins at undesired sites. The recombinant protein was then compared both structurally and functionally to rat plasma apoE. This expression system will be highly useful for probing the ability of rat apoE to mediate food intake in rats.

Animals↗

PubMed2004

Structure of human apolipoprotein A-IV: a distinct domain architecture among exchangeable apolipoproteins with potential functional implications.

Apolipoprotein A-IV (apoA-IV) is an exchangeable apolipoprotein that shares many functional similarities with related apolipoproteins such as apoE and apoA-I but has also been implicated as a circulating satiety factor. However, despite the fact that it contains many predicted amphipathic alpha-helical domains, relatively little is known about its tertiary structure. We hypothesized that apoA-IV exhibits a characteristic functional domain organization that has been proposed to define apoE and apoA-I. To test this, we created truncation mutants in a bacterial system that deleted amino acids from either the N- or C-terminal ends of human apoA-IV. We found that apoA-IV was less stable than apoA-I but was more highly organized in terms of its cooperativity of unfolding. Deletion of the extreme N and C termini of apoA-IV did not significantly affect the cooperativity of unfolding, but deletions past amino acid 333 on the C terminus or amino acid 61 on the N terminus had major destabilizing effects. Functionally, apoA-IV was less efficient than apoA-I at clearing multilamellar phospholipid liposomes and promoting ATP-binding cassette transporter A1-mediated cholesterol efflux. However, deletion of a C-terminal region of apoA-IV, which is devoid of predicted amphipathic alpha helices (amino acids 333-376) stimulated both of these activities dramatically. We conclude that the amphipathic alpha helices in apoA-IV form a single, large domain that may be similar to the N-terminal helical bundle domains of apoA-I and apoE but that apoA-IV lacks the C-terminal lipid-binding and cholesterol efflux-promoting domain present in these apolipoproteins. In fact, the C terminus of apoA-IV appears to reduce the ability of apoA-IV to interact with lipids and promote cholesterol efflux. This indicates that, although apoA-IV may have evolved from gene duplication events of ancestral apolipoproteins and shares the basic amphipathic helical building blocks, the overall localization of functional domains within the sequence is quite different from apoA-I and apoE.

ATP-Binding Cassette Transporters↗

PubMed2003

Expression of biologically active rat apolipoprotein AIV in Escherichia coli.

Rat apolipoprotein AIV (apo AIV) is a 43-kDa intestinal apolipoprotein that is important in lipid metabolism and the suppression of food intake. In this study, a full-length rat apo AIV was expressed in Escherichia coli and purified in a bioactive form. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis revealed that the isolated recombinant protein has a molecular mass of approximately 43 kDa, similar to that of natural rat apo AIV. Immunoblot analysis and N-terminal amino acid sequencing confirmed the identity of the recombinant apo AIV protein as natural rat apo AIV. The recombinant protein was functional in lipoprotein binding assays. Biological activity was assessed behaviorally in that the recombinant protein suppressed food intake of fasted rats comparably to natural rat apo AIV. Neither native nor recombinant apo AIV elicited a conditioned taste aversion (CTA) at doses that suppress feeding. These results indicate that the recombinant apo AIV is structurally and functionally indistinguishable from rat natural apo AIV, making this overexpression and purification scheme a powerful tool for future structure and function studies.

Amino Acid Sequence↗

PubMed2003

The early molecular response to imatinib predicts cytogenetic and clinical outcome in chronic myeloid leukaemia.

Real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) is increasingly used to monitor responses in chronic myeloid leukaemia (CML). The peripheral blood BCR-ABL/ABL ratio, as assessed by Q-RT-PCR, has been shown to correlate with the contemporary cytogenetic response in patients receiving imatinib (Glivec, Gleevec). We have used Q-RT-PCR to monitor the early molecular response to 4 weeks and 3 months of imatinib therapy, in 47 patients with established CML. After 4 weeks of imatinib therapy, patients whose BCR-ABL/ABL ratio had fallen to less than 50% that of baseline had a significantly higher probability of achieving a major cytogenetic response after 6 months of therapy, when compared with those whose ratio did not fall by this amount (P < 0.001). Similarly, patients whose ratio at 3 months was less than 10% of that at baseline had a significantly higher probability of achieving a major cytogenetic remission at 6 months (P < 0.001). Patients who achieved these falls in their BCR-ABL/ABL ratio at either 4 weeks or 3 months had a superior progression-free survival at a median follow-up of 16.5 months (P = 0.01 and 0.003 respectively). These effects were independent of patient age and disease stage. The occurrence of peripheral blood cytopenias sufficiently severe to interrupt therapy was unrelated to progression-free survival. In conclusion, the data suggest that the early trend in the BCR-ABL/ABL ratio may be clinically useful for the early identification of patients destined to fare poorly on imatinib.

Adult↗

PubMed2002

Serial monitoring of BCR-ABL by peripheral blood real-time polymerase chain reaction predicts the marrow cytogenetic response to imatinib mesylate in chronic myeloid leukaemia.

Imatinib mesylate (trade name Glivec or Gleevec) is emerging as an important therapy in the management of chronic myeloid leukaemia (CML). It is clinically useful to monitor the cytogenetic response to imatinib, although frequent marrow examinations are inconvenient. We have used serial real-time reverse transcription-polymerase chain reaction (RT-PCR) to monitor the ratio of peripheral blood BCR-ABL to normal ABL transcripts in 43 patients receiving imatinib, and compared the results to concurrent conventional bone marrow (BM) cytogenetics. After 6 months of treatment, 13 cases were complete cytogenetic responders, defined as all BM metaphases negative for the Philadelphia (Ph) chromosome. In these patients, the BCR-ABL/ABL ratio was less than 0.08%. Six cases achieved a partial cytogenetic response (1-35% Ph-positive BM metaphases) and their BCR-ABL/ABL ratio was between 0.08 and 10%. In total, 24 cases were cytogenetic non-responders, and their BCR-ABL/ABL ratio exceeded 11%. The data suggested that the 6-month BCR-ABL/ABL ratio may reliably predict the contemporary marrow cytogenetic response. It was concluded that serial real-time RT-PCR may offer a convenient surrogate assessment of the marrow cytogenetic response to imatinib therapy in CML.

Adult↗