Daniel Clayton

A genetic linkage map of the bovine genome

A cattle genetic linkage map was constructed which marks about 90% of the expected length of the cattle genome. Over 200 DNA polymorphisms were genotyped in cattle families which comprise 295 individuals in full sibling pedigrees. One hundred and seventy–one loci were found linked to one other locus. Twenty nine of the 30 chromosome pairs are represented by at least one of the 36 linkage groups. Less than a 50 cM difference was found in the male and female genetic maps. The conserved loci on this map show as many differences in gene order compared to humans as is found between humans and mice. The conservation is consistent with the patterns of karyotypic evolution found in the rodents, primates and artiodactyls. This map will be important for localizing quantitative trait loci and provides a basis for further mapping.

The identification of new metallo-β-lactamase inhibitor leads from fragment-based screening.

The emergence of metallo-β-lactamases (MBLs) capable of hydrolysing a broad spectrum of β-lactam antibiotics is particularly concerning for the future treatment of bacterial infections. This work describes the discovery of lead compounds for the development of new inhibitors using a competitive colorimetric assay based on the chromogenic cephalosporin CENTA, and a 500 compound Maybridge™ library suitable for fragment-based screening. The interactions between identified inhibitory fragments and the active site of the MBL from Klebsiella pneumoniae and Pseudomonas aeruginosa were probed by in silico docking studies.

Tyrosine sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3

The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, tyrosine sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential sulfation of two adjacent tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.

A genetic map of DNA loci on bovine chromosome 1

We constructed a genetic map of most of the length of bovine chromosome 1 using the CSIRO and the Texas A&M University cattle reference families. Twelve loci are in a single linkage group, 9 of which are highly polymorphic loci. Four loci are of known biochemical function, alpha-1 crystallin (CRYA1), gamma-s crystallin (CRYG8), superoxide dismutase 1 (SOD1), and uridine monophosphate synthase (UMPS), and these have also been previously mapped in humans. The loci CRYA 1, CSRD 1613, GMBT 7, RM 95, SOD1, and UMPS had been previously assigned to bovine syntenic group U10, while CSRD 1613 and UMPS had also been assigned to chromosome 1 by in situ hybridization. All of the loci show statistically significant linkage to at least one other locus. The conserved loci indicate that there have been major rearrangements during the evolution of bovine chromosome 1 compared to other mammalian chromosomes. The estimate of the total length of the linkage group is 168 cM, which accords well with the predicted length based on chiasmata frequencies for the bovine genome and the relative size of chromosome 1 in the bovine genome.

A Single β-Amino Acid Substitution to Angiotensin II Confers AT 2 Receptor Selectivity and Vascular Function

Novel AT2R ligands were designed by substituting individual &bgr;-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT2R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT1R- or AT2R- transfected HEK-293 cells, only &bgr;-Asp1-Ang II and Ang II fully displaced [125I]-Ang II from AT1R. In contrast, &bgr;-substitutions at each position of Ang II exhibited AT2R affinity, with &bgr;-Tyr4-Ang II and &bgr;-Ile5-Ang II exhibiting ≈1000-fold AT2R selectivity. In mouse aortic rings, &bgr;-Tyr4-Ang II and &bgr;-Ile5-Ang II evoked vasorelaxation that was sensitive to blockade by the AT2R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT1R blockade, &bgr;-Ile5-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (&bgr;-Ile5-Ang II plus candesartan, −24±4 mm Hg) to a greater extent than candesartan alone (−11±3 mm Hg, n=7, P<0.05), an effect that was abolished by concomitant PD123319 infusion. However, in an identical experimental protocol, &bgr;-Tyr4-Ang II had no influence on BP (n=10), and it was less stable than &bgr;-Ile5-Ang II in plasma stability assays. Thus, this study demonstrated that a single &bgr;-amino acid substitution resulted in a compound that demonstrated both in vitro vasorelaxation and in vivo depressor activity via AT2R. This approach to the design and synthesis of novel AT2R-selective peptidomimetics shows great potential to provide insight into AT2R function.

Synthesis of Stapled β3-Peptides through Ring-Closing Metathesis

The first synthesis of carbon-stapled beta(3)-peptides is reported. The precursor beta(3)-peptides, with O-allyl beta-serines located in an i/i+3 relationship, were prepared on solid phase. We show that efficient ring-closing metathesis (RCM) of these new beta(3)-peptides proceeds smoothly either in solution or on an appropriate solid support. All products were generated with high selectivity for the E-isomer.

Structural basis of binding by cyclic nonphosphorylated Peptide antagonists of grb7 implicated in breast cancer progression

Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.

Three‐dimensional NMR structure of the sixth ligand‐binding module of the human LDL receptor: comparison of two adjacent modules with different ligand binding specificities

The sixth ligand‐binding module of the low‐density lipoprotein receptor contributes to the binding of apolipoprotein B100‐containing lipoproteins. 1H NMR spectroscopy, DYANA and X‐PLOR structure calculations were used to determine that this module has a well defined structure with a backbone conformation similar to other modules. Structures from calculations that simulated the presence of a calcium ion showed increased resolution without large increases in energy, increased deviations from idealised geometry or violations of experimental constraints. Investigation of the surface properties of this module indicates there are significant differences from the fifth module, which binds apolipoprotein E‐containing lipoproteins in addition to apolipoprotein B100‐containing lipoproteins.

Uptake of a cell permeable G7-18NATE construct into cells and binding with the Grb7-SH2 domain

Grb7 is an adapter protein found to be overexpressed in several breast and other cancer cell types along with ErbB2. Grb7 is normally an interaction partner with focal adhesion kinase and in cancer cells also aberrantly interacts with ErbB2. It is thus implicated in the migratory and proliferative potential of cancer cells. Previous studies have shown that the phage display‐derived cyclic nonphosphorylated inhibitor peptide, G7‐18NATE, when linked to Penetratin©, is able to interfere with the interaction of Grb7 with its upstream binding partners and to impact on both cell migration and proliferation. Here we report the synthesis of a biotinylated G7‐18NATE covalently attached to just the last seven residues of Penetratin© (G7‐18NATE‐P‐Biotin). We demonstrate that this construct is taken up efficiently into MDA‐MB‐468 breast cancer cells and colocalizes with Grb7 in the cytoplasm. We also used isothermal titration calorimetry to determine the binding affinity of G7‐18NATE‐P‐Biotin to the Grb7‐SH2 domain, and showed that it binds with micromolar affinity (K d = 14.4 μM), similar to the affinity of G7‐18NATE (K d = 35.4 μM). Together this shows that this shorter G7‐18NATE‐P‐Biotin construct is suitable for further studies of the antiproliferative and antimigratory potential of this inhibitor.

Design and Synthesis of Truncated EGF-A Peptides that Restore LDL-R Recycling in the Presence of PCSK9 In Vitro

Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 μM) compared with wild-type EGF-A (KD = 1.2 μM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.