Characterization and properties of dominant-negative mutants of the ras-specific guanine nucleotide exchange factor CDC25(Mm).

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation depends on the competing action of GTPase activating proteins and guanine nucleotide exchange factors (GEF). The properties of two dominant-negative mutants within the catalytic domains of the ras-specific GEF, CDC25(Mm), are described. In vitro, the mutant GEF(W1056E) and GEF(T1184E) proteins are catalytically inactive, are able to efficiently displace wild-type GEF from p21(ras), and strongly reduce affinity of the nucleotide-free ras x GEF complex for the incoming nucleotide, thus resulting in the formation of a stable ras.GEF binary complex. Consistent with their in vitro properties, the two mutant GEFs bring about a dramatic reduction in ras-dependent fos-luciferase activity in mouse fibroblasts. The stable ectopic expression of the GEF(W1056E) mutant in smooth muscle cells effectively reduced growth rate and DNA synthesis with no detectable morphological changes.

Inhibitory activity of IL-1 receptor antagonist depends on the balance between binding capacity for IL-1 receptor type 1 and IL-1 receptor type II.

A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.

Transfected type II interleukin-1 receptor impairs responsiveness of human keratinocytes to interleukin-1.

Of the two known types of specific receptors for interleukin (IL)-1, the function of the type II IL-1 receptor (IL-1RII) is still elusive. IL-1RII is allegedly devoid of signaling capacity and is therefore thought to act by trapping and inhibiting IL-1. To directly assess the functional role of IL-1RII, a human keratinocyte cell line has been stably transfected with a cDNA coding for IL-1RII, and its responsiveness to IL-1 has been compared with that of nontransfected cells. Parental cells express IL-1RI and are responsive to low doses of IL-1, whereas transfected cells overexpress IL-1RII, both in its membrane and soluble form, and show a dramatically impaired response to IL-1. Selective block of IL-1RII restores the ability of transfected keratinocytes to respond to IL-1, indicating that the overexpressed IL-1RII is in fact uniquely responsible for their refractoriness to IL-1. The main mechanism of unresponsiveness in transfected keratinocytes appears to be the capture and neutralization of IL-1 by the soluble form of IL-1RII.

Heavy-metal modulation of the human intercellular adhesion molecule (ICAM-1) gene expression.

The intercellular adhesion molecule 1 (ICAM-1) can be induced on many different cell types by a set of various modulators (IL1 beta, TNF, LPS, IFN-gamma), which are released during the inflammatory process. We have investigated the possibility that other factors, related to the stress and biophysical perturbations of the inflammatory response, may also modulate ICAM-1. Here, we report that heavy metals, in particular zinc, can enhance the expression of the ICAM-1 gene on cells actively involved at different levels during inflammation. Kinetic studies of ICAM-1 gene expression shows a maximum level of induction 4 h after treatment with metals, followed by a rapid decrease to basal levels within 12 h. The effect on enhanced gene expression is mostly due to a rapid increase of the transcriptional rate as shown by nuclear run-on experiments. In B lymphoblastoid cells, but not in fibroblasts, the increase in RNA expression seems significantly greater that the subsequent increase in protein expression, suggesting that a further point of post-transcriptional regulation of ICAM-1 occurs and may be linked to the cellular specificity. may be linked to the cellular specificity.