Target-directed enediynes: designed estramycins.

The goal of selective targeting of enediyne cytotoxins has been investigated using estrogenic delivery vehicles. A series of estrogen-enediyne conjugates were assembled, and affinity for human estrogen receptor [hERalpha] was determined. The most promising candidate induced receptor degradation following Bergman cycloaromatization and caused inhibition of estrogen-induced transcription in T47-D human breast cancer cells.

Protein-degrading enediynes: library screening of Bergman cycloaromatization products.

[structure: see text] A screening method based on Bergman cycloaromatization products was applied to a compact library of estrogenic-enediyne hybrids. An enediyne candidate identified from the screen was subsequently synthesized, and it induced temperature- and concentration-dependent degradation of human estrogen receptor alpha upon cycloaromatization.

Functional analysis of a novel estrogen receptor-beta isoform.

A new level of complexity has recently been added to estrogen signaling with the identification of a second estrogen receptor, ERbeta. By screening a rat prostate cDNA library, we detected ERbeta as well as a novel isoform that we termed ERbeta2. ERbeta2 contains an in-frame inserted exon of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ERbeta hormone-binding domain. We also have evidence for the expression of both ERbeta1 and ERbeta2 in human cell lines. Competition ligand binding analysis of bacterially expressed fusion proteins revealed an 8-fold lower affinity of ERbeta2 for 17beta-estradiol (E2) [dissociation constant (Kd approximately 8 nM)] as compared with ERbeta1 (Kd approximately 1 nM). In vitro transcribed and translated ERbeta1 and ERbeta2 bind specifically to a consensus estrogen responsive element in a gel mobility shift assay. Furthermore, we show heterodimerization of ERbeta1 and ERbeta2 with each other as well as with ERalpha. In affinity interaction assays for proteins that associate specifically with the hormone-binding domain of these receptors, we demonstrate that the steroid receptor coactivator SRC-1 interacts in an estrogen-dependent manner with ERalpha and ERbeta1, but not with ERbeta2. In cotransfection experiments with expression plasmids for ERalpha, ERbeta1, and ERbeta2 and an estrogen-responsive element-containing luciferase reporter, the dose response of ERbeta1 to E2 was similar to that of ERalpha although the maximal stimulation was approximately 50%. In contrast, ERbeta2 required 100- to 1000-fold greater E2 concentrations for maximal activation. Thus, ERbeta2 adds yet another facet to the possible cellular responses to estrogen.

Translational and pretranslational regulation of protein synthesis by amino acid availability in primary cultures of rat hepatocytes.

Protein synthesis is inhibited in both rat liver and isolated rat hepatocytes following deprivation of single essential amino acids. The aim of the present study was to define the time course of changes in peptide-chain initiation, albumin synthesis, and albumin mRNA following histidine deprivation and the reversal of these changes in response to readdition of the deprived amino acid. A further aim was to ascertain whether there was an accommodation of the inhibition of initiation following long-term amino acid deprivation. Primary cultures of rat hepatocytes were maintained in serum-free medium containing either all amino acids (complete medium) or all except histidine. Synthesis of total protein was reduced to 34% of control values following 48 hr of histidine deprivation and was restored to control values within 1 hr of addition of complete medium to histidine-deprived cells. These changes in protein synthesis were due to translational regulation involving initiation. No accommodation of the inhibition was observed following long-term deprivation of histidine as has been observed under other conditions of cellular stress. The synthesis of albumin was reduced to a greater extent than that of total protein, and required 72 hr to recover to control values following return to complete medium. These changes in albumin synthesis were due to a combination of altered initiation and a mechanism involving pretranslational regulation as evidenced by corresponding alterations in albumin mRNA. The results show that amino acid availability controls protein synthesis in liver cells through both translational and pretranslational mechanisms.