Biomedical subjects

K H Bremner

Publications and source records attributed to K H Bremner.

3 recordsLinked to original sources

PubMed2001

Structural determinants outside the PXDLS sequence affect the interaction of adenovirus E1A, C-terminal interacting protein and Drosophila repressors with C-terminal binding protein.

C-Terminal binding protein (CtBP) interacts with a highly conserved amino acid motif (PXDLS) at the C terminus of adenovirus early region 1A (AdE1A) protein. This amino acid sequence has recently been demonstrated in the mammalian protein C-terminal interacting protein (CtIP) and a number of Drosophila repressors including Snail, Knirps and Hairy. In the study described here we have examined the structures of synthetic peptides identical to the CtBP binding sites on these proteins using NMR spectroscopy. It has been shown that peptides identical to the CtBP binding site in CtIP and at the N terminus of Snail form a series of beta-turns similar to those seen in AdE1A. The PXDLS motif towards the C terminus of Snail forms an alpha-helix. However, the motifs in Knirps and Hairy did not adopt well-defined structures in TFE/water mixtures as shown by the absence of medium range NOEs and a high proportion of signal overlap. The affinities of peptides for Drosophila and mammalian CtBP were compared using enzyme-linked immunosorbent assay. CtIP, Snail (N-terminal peptide) and Knirps peptides all bind to mammalian CtBP with high affinity (K(i) of 1.04, 1.34 and 0.52 microM, respectively). However, different effects were observed with dCtBP, most notably the affinity for the Snail (N-terminal peptide) and Knirps peptides were markedly reduced (K(i) of 332 and 56 microM, respectively) whilst the Hairy peptide bound much more strongly (K(i) for dCtBP of 6.22 compared to 133 microM for hCtBP). In addition we have shown that peptides containing identical PXDLS motifs but with different N and C terminal sequences have appreciably different affinities for mammalian CtBP and different structures in solution. We conclude that the factors governing the interactions of CtBPs with partner proteins are more complex than simple possession of the PXDLS motif. In particular the overall secondary structures and amino acid side chains in the binding sites of partner proteins are of importance as well as possible global structural effects in both members of the complex. These data are considered evidence for a multiplicity of CtBPs and partner proteins in the cell.

Adenovirus E1A Proteins↗

PubMed2001

Harnessing nuclear localization pathways for transgene delivery.

Inefficient transport of DNA from the cytoplasm into the nucleus remains a limiting step in the development of non-viral gene delivery systems. This is particularly acute in non-dividing cells, where entry to the nucleus is thought to occur only through the nuclear pore complex. Active import of physiological proteins is mediated by nuclear localization sequences (NLSs) within cargo proteins such as transcription factors. Here we review current knowledge of this import machinery and consider its exploitation by mammalian viruses. Significant research effort has been directed at incorporating NLSs into synthetic gene delivery systems to take advantage of this physiological pathway. Both non-covalent and covalent methods of conjugation are evaluated, with NLS linkage to both DNA and carrier, and compared with activities of simple cationic polymers. Finally, progress in the field of DNA sequence-specific nuclear import is examined and the current state of the technology assessed.

Active Transport, Cell Nucleus↗

PubMed2000

Structural determinants in adenovirus 12 E1A involved in the interaction with C-terminal binding protein 1.

The interaction between the C-terminal binding protein 1 (CtBP-1) and purified Ad12 E1A protein has been examined through the use of a combination of biophysical techniques. A fragment equivalent to the 77 C-terminal amino acids of Ad12 E1A (Ad12 77-a.a. E1A) was generated by limited proteolysis of Ad12 266-a.a. E1A at Phe(187) and/or Tyr(189) using chymotrypsin. The impact of deletion of the 189 N-terminal amino acids from E1A on the equilibrium dissociation constant K(d) for binding to CtBP was assessed using ELISA in vitro binding assays and intrinsic fluorescence spectroscopy. Values of K(d) of 4.0 and 38 nM were determined for full-length and truncated forms of E1A, respectively. Circular dichroism spectroscopic studies revealed that the conformation adopted by these polypeptides is dependent on the surrounding environment, which is predominately randomly folded when free in solution, but adopting a more ordered alpha-helical secondary structure in the presence of trifluoroethanol. Using nuclear magnetic resonance (NMR) spectroscopy to examine the interaction between Ad E1A and CtBP it was observed that the chemical shift positions of individual backbone amide nitrogen atoms were well resolved in (15)N-(1)H-HSQC NMR spectra performed on samples of isotopically (15)N-labeled Ad12 77-a.a. E1A. In the presence of CtBP, signals of backbone amide nitrogen atoms displayed increased linewidth consistent with an increase in molecular mass upon binding CtBP. In addition, some signals that have been attributed to Val(254/256) and Leu(259), and reside within the binding site for CtBP on E1A, are shifted in the (15)N- and/or (1)H-dimensions, defining specific contacts between E1A and CtBP. These data suggest that structural determinants in the C-terminal PXDLS binding motif in the rest of exon 2 and in exon 1 all contribute to optimizing the conformation of the binding site on Ad12 E1A for CtBP. However, no interaction was observed between CtBP and truncated Ad12 E1A, which no longer contained the C-terminal binding motif.

Adenovirus E1A Proteins↗