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Richard D Brokx

Publications and source records attributed to Richard D Brokx.

5 recordsLinked to original sources
PubMed2003

Nuclear magnetic resonance-based dissection of a glycosyltransferase specificity for the mucin MUC1 tandem repeat.

The human glycoprotein MUC1 mucin plays a critical role in cancer progression. Breast, ovarian, and colon cancer cells often display unique cell-surface antigens corresponding to aberrantly glycosylated forms of the MUC1 tandem repeat. In this report, (15)N- and (13)C-labeled forms of a recombinant MUC1 construct containing five tandem repeats were used as substrates to define the order and kinetics of addition of N-acetylgalactosamine (GalNAc) moieties by a recombinant active form of the human enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGalNAc-T1; residues 40-559). Heteronuclear NMR experiments were performed to assign resonances associated with the two serines (Ser5 and Ser15) and three threonines (Thr6, Thr14, and Thr19) present in the 20-residue long MUC1 repeat. The kinetics and order of addition of GalNAc moieties (Tn antigen) on the MUC1 construct by human ppGalNAc-T1 were subsequently dissected by NMR spectroscopy. Threonine 14 was shown to be rapidly glycosylated by ppGalNAc-T1 with an initial rate of 25 microM/min, followed by Thr6 (8.6 microM/min). The enzyme also modified Ser5 at a slower rate (1.7 microM/min), an event that started only after the glycosylation of Thr14 and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified by ppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This study demonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominant substrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. More importantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relative simplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylation pathways and the rational design of tumor-associated MUC1 antigens.

Acetylgalactosamine↗
PubMed2002

A stable human p53 heterotetramer based on constructive charge interactions within the tetramerization domain.

The human p53 tetramerization domain (called p53tet; residues 325-355) spontaneously forms a dimer of dimers in solution. Hydrophobic interactions play a major role in stabilizing the p53 tetramer. However, the distinctive arrangement of charged residues at the dimer-dimer interface suggests that they also contribute to tetramer stability. Charge-reversal mutations at positions 343, 346, and 351 within the dimer-dimer interface were thus introduced into p53tet constructs and shown to result in the selective formation of a stable heterotetramer composed of homodimers. More precisely, mutants p53tet-E343K/E346K and p53tet-K351E preferentially associated with each other, but not with wild-type p53tet, to form a heterodimeric tetramer with enhanced thermal stability relative to either of the two components in isolation. The p53tet-E343K/E346K mutant alone assembled into a weakly stable tetramer in solution, whereas p53tet-K351E existed only as a dimer. Moreover, these mutants did not form heterocomplexes with wild-type p53tet, illustrating the specificity of the ionic interactions that form the novel heterotetramer. This study demonstrates the dramatic importance of ionic interactions in altering the stability of the p53 tetramer and in selectively creating heterotetramers of this protein scaffold.

Amino Acid Sequence↗
PubMed2002

Designing peptide-based scaffolds as drug delivery vehicles.

Methods for delivering drugs into cells remain an important part of the process of designing drugs. One promising approach is the concept of loligomers, synthetic peptides composed of a branched polylysine core harboring identical arms. Loligomers are typically synthesized with eight arms, each carrying peptide signals guiding their import and localization into cells. The most important advantage of loligomers is the multivalent presentation of targeting signals resulting from a tentacular arrangement. Multivalency increases the efficiency of import and intracellular routing signals as compared to similar linear peptides. Secondly, it reduces and delays the impact of peptide degradation in terms of cellular processing and compartmentalization. The vectorial delivery of nucleus-directed loligomers into cells has recently been confirmed by microscopy and flow cytometry studies. Practical uses of loligomers as intracellular vehicles include the import of plasmid DNA into cells, the conjugation of chemical groups, such as photosensitizers for use in photodynamic therapy, and the incorporation of cytotoxic T-lymphocyte (CTL) epitopes with a view to creating synthetic vaccines. Branched peptides such as loligomers represent simple and versatile molecular vehicles with potential applications in a wide variety of drug design approaches.

Drug Delivery Systems↗