Biomedical subjects

Timothy R Dafforn

Publications and source records attributed to Timothy R Dafforn.

24 records · Page 2Linked to original sources

PubMed2003

Targeting a surface cavity of alpha 1-antitrypsin to prevent conformational disease.

Conformational diseases are caused by a structural rearrangement within a protein that results in aberrant intermolecular linkage and tissue deposition. This is typified by the polymers that form with the Z deficiency variant of alpha 1-antitrypsin (Glu-342 --> Lys). These polymers are retained within hepatocytes to form inclusions that are associated with hepatitis, cirrhosis, and hepatocellular carcinoma. We have assessed a surface hydrophobic cavity in alpha1-antitrypsin as a potential target for rational drug design in order to prevent polymer formation and the associated liver disease. The introduction of either Thr-114 --> Phe or Gly-117 --> Phe on strand 2 of beta-sheet A within this cavity significantly raised the melting temperature and retarded polymer formation. Conversely, Leu-100 --> Phe on helix D accelerated polymer formation, but this effect was abrogated by the addition of Thr-114 --> Phe. None of these mutations affected the inhibitory activity of alpha 1-antitrypsin. The importance of these observations was underscored by the finding that the Thr-114 --> Phe mutation reduced polymer formation and increased the secretion of Z alpha 1-antitrypsin from a Xenopus oocyte expression system. Moreover cysteine mutants within the hydrophobic pocket were able to bind a range of fluorophores illustrating the accessibility of the cavity to external agents. These results demonstrate the importance of this cavity as a site for drug design to ameliorate polymerization and prevent the associated conformational disease.

Animals↗

PubMed2003

Murine serpin 2A is a redox-sensitive intracellular protein.

Murine serpin 2A is expressed at high levels in haemopoietic progenitors and down-regulated on differentiation. When it is constitutively expressed in the multipotent haemopoietic cell line, FDCP-Mix, it causes a delay in differentiation and increased clonogenic potential. The serpin is also dramatically up-regulated on T-cell activation. It has an unusual reactive site Cys-Cys sequence, a unique C-terminal extension and lacks a typical cleavable N-terminal signal sequence. In spite of these features, the protein is not a member of the ovalbumin-serpin family, but is instead most closely related to human antichymotrypsin. We have shown that the serpin is intracellular with prominent nuclear localization. Transverse urea gradient gels and CD studies show that the protein undergoes the stressed-relaxed conformational change typical of inhibitory serpins. However, we have not detected complex-forming activity with a set of proteases. Thermal denaturation studies also show that the protein has decreased structural stability under reducing conditions, although it lacks disulphide bonds within the core of the molecule. Our results show that serpin 2A is an intracellular protein with the potential to mediate its biological effects via interaction with non-protease intracellular targets. Furthermore, the results presented suggest a model whereby the serpin interactions could be modulated by redox conditions or conformational change induced by cleavage of the reactive-site loop.

Animals↗

PubMed2003

Nucleation of alpha 1-antichymotrypsin polymerization.

Alpha(1)-antichymotrypsin is an acute phase plasma protein and a member of the serpin superfamily. We show here that wildtype alpha(1)-antichymotrypsin forms polymers between the reactive center loop of one molecule and the beta-sheet A of a second at a rate that is dependent on protein concentration and the temperature of the reaction. The rate of polymerization was accelerated by seeding with polymers of alpha(1)-antichymotrypsin and a complex of alpha(1)-antichymotrypsin with an exogenous reactive loop peptide but not with reactive loop cleaved alpha(1)-antichymotrypsin or with polymers of other members of the serpin superfamily. Sonication of alpha(1)-antichymotrypsin polymers markedly increased the efficacy of seeding such that polymers were able to form under physiological conditions. Taken together, these data provide the first demonstration that serpin polymerization can result from seeding. This mechanism is analogous to the fibrillization of the Abeta(1-42) peptide and may be important in the deposition of alpha(1)-antichymotrypsin in the plaques of Alzheimer's disease.

Antithrombins↗

PubMed2003

Drosophila necrotic mutations mirror disease-associated variants of human serpins.

Polymerization of members of the serpin superfamily underlies diseases as diverse as cirrhosis, angioedema, thrombosis and dementia. The Drosophila serpin Necrotic controls the innate immune response and is homologous to human alpha(1)-antitrypsin. We show that necrotic mutations that are identical to the Z-deficiency variant of alpha(1)-antitrypsin form urea-stable polymers in vivo. These necrotic mutations are temperature sensitive, which is in keeping with the temperature-dependent polymerization of serpins in vitro and the role of childhood fevers in exacerbating liver disease in Z alpha-antitrypsin deficiency. In addition, we identify two nec mutations homologous to an antithrombin point mutation that is responsible for neonatal thrombosis. Transgenic flies carrying an S>F amino-acid substitution equivalent to that found in Siiyama-variant antitrypsin (nec(S>F.UAS)) fail to complement nec-null mutations and demonstrate a dominant temperature-dependent inactivation of the wild-type nec allele. Taken together, these data establish Drosophila as a powerful system to study serpin polymerization in vivo.

Animals↗

PubMed2002

Characterization of the necrotic protein that regulates the Toll-mediated immune response in Drosophila.

Necrotic (Nec) is an important component of the proteolytic cascade that activates the Toll-mediated immune response in Drosophila. The Nec protein is a member of the serpin (SERine Protease INhibitor) superfamily and is thought to regulate the cascade by inhibiting the serine protease Persephone. Nec was expressed in Escherichia coli, and the purified protein folded to the active native conformation required for protease inhibitory activity. Biochemical analysis showed that Nec had a broad inhibitory specificity and inhibited elastase, thrombin, and chymotrypsin-like proteases. It did not inhibit trypsin or kallikrein. These data show that Necrotic is likely to inhibit a wide range of proteases in Drosophila and that Nec has the specificity requirements to act as the physiological inhibitor of Persephone in vivo.

Animals↗

PubMed2001

6-mer peptide selectively anneals to a pathogenic serpin conformation and blocks polymerization. Implications for the prevention of Z alpha(1)-antitrypsin-related cirrhosis.

Conformational diseases such as amyloidosis, Alzheimer's disease, prion diseases, and the serpinopathies are all caused by structural rearrangements within a protein that transform it into a pathological species. These diseases are typified by the Z variant of alpha(1)-antitrypsin (E342K), which causes the retention of protein within hepatocytes as inclusion bodies that are associated with neonatal hepatitis and cirrhosis. The inclusion bodies result from the Z mutation perturbing the conformation of the protein, which facilitates a sequential interaction between the reactive center loop of one molecule and beta-sheet A of a second. Therapies to prevent liver disease must block this reactive loop-beta-sheet polymerization without interfering with other proteins of similar tertiary structure. We have used reactive loop peptides to explore the differences between the pathogenic Z and normal M alpha(1)-antitrypsin. The results show that the reactive loop is likely to be partially inserted into beta-sheet A in Z alpha(1)-antitrypsin. This conformational difference from M alpha(1)-antitrypsin was exploited with a 6-mer reactive loop peptide (FLEAIG) that selectively and stably bound Z alpha(1)-antitrypsin. The importance of this finding is that the peptide prevented the polymerization of Z alpha(1)-antitrypsin and did not significantly anneal to other proteins (such as antithrombin, alpha(1)-antichymotrypsin, and plasminogen activator inhibitor-1) with a similar tertiary structure. These findings provide a lead compound for the development of small molecule inhibitors that can be used to treat patients with Z alpha(1)-antitrypsin deficiency. Furthermore they demonstrate how a conformational disease process can be selectively inhibited with a small peptide.

Acrylamide↗