Background

The present study was designed to determine whether some of the effects of estrogen on human vascular cell growth are exerted through membrane-binding sites, using native as well as novel protein-bound, membrane non-permeant estrogenic complexes. We measured changes in DNA synthesis and creatine kinase-specific activity (CK), after treatment with estradiol-17beta (E(2)), estradiol-17beta-6-(O)-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (E(2)-BSA), 6-carboxymethyl genistein (CG) or 6- carboxymethyl genistein bound to the high molecular protein keyhole limpet hemocyanin (CG-KLH), and 7-(O)-carboxymethyl daidzein (CD) or 7-(O)-carboxymethyl daidzein linked to keyhole limpet hemocyanin (CD-KLH). High concentrations of either E(2) or E(2)-BSA inhibited DNA synthesis in vascular smooth muscle cells (VSMC) (-39% +/- 28% v -32% +/- 15%). Estradiol as well as CG and CD increased DNA synthesis dose dependently in endothelial ECV-304 cells. The CG and CD, as well as CG-KLH and CD-KLH, stimulated DNA synthesis dose dependently in VSMC (66% +/- 2%, 100% +/- 12%, 66% +/- 6%, and 41% +/- 8% at 300 nmol/L, respectively). In contrast all forms of protein-bound hormones were unable to affect DNA synthesis in ECV-304 cells or CK in either cell type. In VSMC, both free and bound hormones increased mitogen-activated protein-kinase (MAPK)-kinase activity, which was blocked by UO126, an inhibitor of MAPK-kinase. Furthermore, the effects of E(2), E(2)-BSA, or CG-KLH on DNA synthesis were inhibited by UO126. Using the E(2)-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for E(2) in VSMC and ECV 304 cells. Hence, the effects of E(2) on DNA synthesis in human VSMC, but not in endothelial cells, are apparently exerted by membrane-binding sites for E(2) and do not require intracellular entry of E(2) through the classic nuclear receptor route.

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono-and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.Targeting of most substrates to the 26 S proteasome requires covalent marking with polyubiquitin chains. Protein ubiquitination is a multistep process accomplished by the concerted action of three enzymes. The reaction begins with the ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin and then forms a thioester bond between the activated glycine residue and a cysteine residue in its active site. Subsequently, a ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a trans-thioesterification reaction. Finally, a ubiquitin-protein ligase (E3) recruits a target protein and guides the transfer of the activated ubiquitin from the E2 to the substrate (1-3). Ubiquitin transfer from the E2 enzyme to the substrate is catalyzed directly by really interesting new gene (RING) 3 finger-containing E3s or indirectly when a homologous to E6-AP C terminus (HECT) domain E3 is mediating the transfer (4). Several forms of ubiquitination have been identified (5). Monoubiquitination or multiple monoubiquitinations are referred to as the conjugation of single or multiple ubiquitin moieties to distinct lysine residues on the substrate. These forms of ubiquitination were implicated in various cellular pathways, which include endocytosis and sorting of proteins to different cellular compartments (6, 7), as well as in several cases of proteasomal activity, such as the processing of the p105 precursor of the transcription regulator NF-B (8). However, polyubiquitination is the most common post-translational modification of proteins destined for degradation (9).In polyubiquitination assembly, ubiquitin conjugation was originally thought to be repeated in a cyclic manner whereby in each step a new moiety of ubiquitin is linked to one of the lysine residues of the previously conjugated ubiquitin. However, in view of recent findings, several alternative mechanisms have been proposed (10). Li et al. (11) demonstrated in a recon...