No abstract

MetJ is a member of the ribbon-helix-helix class of DNA-binding proteins whose a⁄nity for operators is apparently controlled by an unprecedented long-range electrostatic e¡ect from the tertiary sulphur atom of its co-repressor, S-adenosyl methionine. We report here the results of kinetic assays of DNA binding with MetJ mutant proteins having altered net charges. The results (a) suggest that MetJ locates its operators via a sliding mechanism, (b) support the idea that electrostatic steering is important in the initial DNA binding event and (c) highlight the sensitivity of this system to electrostatic e¡ects.

Eph receptor tyrosine kinases and ephrins regulate morphogenesis in the developing embryo where they effect adhesion and motility of interacting cells. Although scarcely expressed in adult tissues, Eph receptors and ephrins are overexpressed in a range of tumours. In malignant melanoma, increased Eph and ephrin expression levels correlate with metastatic progression. We have examined cellular and biochemical responses of EphA3-expressing melanoma cell lines and human epithelial kidney 293T cells to stimulation with polymeric ephrin-A5 in solution and with surfaces of defined ephrin-A5 densities. Within minutes, rapid reorganisation of the actin and myosin cytoskeleton occurs through activation of RhoA, leading to the retraction of cellular protrusions,membrane blebbing and detachment, but not apoptosis. These responses are inhibited by monomeric ephrin-A5, showing that receptor clustering is required for this EphA3 response. Furthermore, the adapter CrkII, which associates with tyrosine-phosphorylated EphA3 in vitro, is recruited in vivo to ephrin-A5-stimulated EphA3. Expression of an SH3-domain mutated CrkII ablates cell rounding, blebbing and detachment. Our results suggest that recruitment of CrkII and activation of Rho signalling are responsible for EphA3-mediated cell rounding, blebbing and de-adhesion, and that ephrin-A5-mediated receptor clustering and EphA3 tyrosine kinase activity are essential for this response.

Serpins inhibit cognate serine proteases involved in a number of important processes including blood coagulation and inflammation. Consequently, loss of serpin function or stability results in a number of disease states. Many of the naturally occurring mutations leading to disease are located within strand 1 of the C ␤-sheet of the serpin. To ascertain the structural and functional importance of each residue in this strand, which constitutes the so-called distal hinge of the reactive center loop of the serpin, an alanine scanning study was carried out on recombinant ␣ 1 -antitrypsin Pittsburgh mutant (P1 ‫ס‬ Arg). Mutation of the P10Ј position had no effect on its inhibitory properties towards thrombin. Mutations to residues P7Ј and P9Ј caused these serpins to have an increased tendency to act as substrates rather than inhibitors, while mutations at P6Ј and P8Ј positions caused the serpin to behave almost entirely as a substrate. Mutations at the P6Ј and P8Ј residues of the C ␤-sheet, which are buried in the hydrophobic core in the native structure, caused the serpin to become highly unstable and polymerize much more readily. Thus, P6Ј and P8Ј mutants of ␣ 1 -antitrypsin had melting temperatures 14 degrees lower than wild-type ␣ 1 -antitrypsin. These results indicate the importance of maintaining the anchoring of the distal hinge to both the inhibitory mechanism and stability of serpins, the inhibitory mechanism being particularly sensitive to any perturbations in this region. The results of this study allow more informed analysis of the effects of mutations found at these positions in disease-associated serpin variants.