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Biomedical subjects

John D Norris

Publications and source records attributed to John D Norris.

8 recordsLinked to original sources
PubMed2005

Single-step purification of full-length human androgen receptor.

The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 microM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions. The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.

Journal Article↗
PubMed2005

Structural basis for an unexpected mode of SERM-mediated ER antagonism.

Tamoxifen is effective for the prevention and treatment of estrogen-dependent breast cancers, but is associated with an increased incidence of endometrial tumors. We report the crystal structure of the estrogen receptor alpha (ERalpha) ligand binding domain (LBD) bound to the structurally similar compound GW5638, which has therapeutic potential and does not stimulate the uterus. Like tamoxifen, GW5638 relocates the carboxy-terminal helix (H12) to the known coactivator-docking site in the ERalpha LBD. However, GW5638 repositions residues in H12 through specific contacts with the N terminus of this helix. In contrast to tamoxifen, the resulting increase in exposed hydrophobic surface of ERalpha LBD correlates with a significant destabilization of ERalpha in MCF-7 cells. Thus, the GW5638-ERalpha LBD structure reveals an unexpected mode of SERM-mediated ER antagonism, in which the stability of ERalpha is decreased through an altered position of H12. This dual mechanism of antagonism may explain why GW5638 can inhibit tamoxifen-resistant breast tumors.

Amino Acid Sequence↗
PubMed2002

Elucidation of the molecular mechanism of action of selective estrogen receptor modulators.

The term selective estrogen receptor modulator (SERM) describes a group of pharmaceuticals that manifest estrogen receptor (ER) agonist activity in some tissues but opposes estrogen action in others. Although the name describing this class of drugs is new, the concept is not, as compounds exhibiting tissue-selective ER agonist/antagonist properties have been available for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of SERMs and develop them as target organ-selective ER agonists for the treatment of osteoporosis and other estrogenopathies. This realization has provided the impetus for research in this area, the progress of which is described in this review.

Estradiol↗
PubMed2002

Connections and regulation of the human estrogen receptor.

Estrogen regulates a plethora of functionally dissimilar processes in a broad range of tissues. Recent progress in the study of the molecular mechanism of action of estrogen(s) has revealed why different cells can respond to the same hormone in a different manner. Three of these findings are of particular importance: (i) There are two genetically and functionally distinct estrogen receptors that have distinct expression patterns in vivo; (ii) the positive and negative transcriptional activities of these receptors require them to engage transcription cofactors (coactivators or corepressors) in target cells; and (iii) not all cofactors are functionally equivalent, nor are they expressed in the same manner in all cells. Thus, although the estrogen receptor is required for a cell to respond to an estrogenic stimulus, the nature and extent of that response are determined by the proteins, pathways, and processes with which the receptor interacts.

Acetyltransferases↗
PubMed2002

Identification of a negative regulatory surface within estrogen receptor alpha provides evidence in support of a role for corepressors in regulating cellular responses to agonists and antagonists.

Several lines of evidence have indicated that the estrogen receptor (ER) can recruit the corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid receptors (SMRT), to target genes in the presence of tamoxifen, suggesting a possible role for NCoR/SMRT in regulating ER pharmacology. However, a tamoxifen-dependent, direct interaction between NCoR/SMRT and ER in vitro has not been demonstrated. To investigate the possible involvement of different corepressors in the actions of antiestrogen-bound ER, we have constructed a phage display library that expresses 23-amino acid peptides containing the canonical CoRNR box motif in an otherwise random background. Screening of the CoRNR box library with apo-ER or ER treated with tamoxifen or ICI 182,780 led to the isolation of peptides whose ability to interact with ER was influenced by the nature of the bound ligand. Using a series of ERalpha mutants, we found that helix 12 was not required for the binding of CoRNR box peptides, whereas disruption of helixes 3 and 5 had a marked effect on peptide binding. One mutant, ER-L372R, lost the ability to interact with CoRNR box-containing peptides without affecting its binding to LXXLL motif-containing peptides. The estradiol- and tamoxifen-mediated transcriptional activity of ER-L372R was dramatically increased by 11- and 3-fold, respectively, compared with that of wild-type ERalpha. The ICI 182,780-mediated repressional activity of this mutant was also reduced by 4-fold compared with that of wild-type ERalpha. These results suggest that leucine 372 may be an important part of the interaction surface on ER that is responsible for corepressor binding. In addition, our data suggest that corepressors, other than NCoR/SMRT, may be involved in ER signaling.

Amino Acid Motifs↗
PubMed2002

A negative coregulator for the human ER.

ERalpha is a ligand-activated transcription factor and a key regulator of the processes involved in cellular proliferation and differentiation. In addition, aberrant ERalpha activity is linked to several pathological conditions including breast cancer. A complex network of coregulatory proteins is largely believed to determine the transcriptional activity of ERalpha. We report here the isolation of a protein, denoted RTA for repressor of tamoxifen transcriptional activity, which contains an RNA recognition motif and interacts with the receptor N-terminal activation domain. RTA interacts with RNA in vitro, and its overexpression inhibits the partial agonist activity manifest by the antiestrogen tamoxifen while minimally affecting E2-activated transcription. Mutation of the RNA recognition motif alters RNA binding specificity and results in a dominant negative form of RTA that leads to derepression of ERalpha transcriptional activity, allowing all classes of antiestrogens to manifest partial agonist activity and enhancing agonist efficacy. These findings suggest a role for RNA binding proteins as coregulatory factors of the nuclear receptor family and reveal a novel mechanism by which antiestrogens can manifest agonist activities in some tissues.

Amino Acid Sequence↗
PubMed2002

Definition of the molecular and cellular mechanisms underlying the tissue-selective agonist/antagonist activities of selective estrogen receptor modulators.

The term selective estrogen receptor modulators describes a group of pharmaceuticals that function as estrogen receptor (ER) agonists in some tissues but that oppose estrogen action in others. Although the name for this class of drugs has been adopted only recently, the concept is not new, as compounds exhibiting tissue-selective ER agonist/antagonist properties have been around for nearly 40 years. What is new is the idea that it may be possible to capitalize on the paradoxical activities of these drugs and develop them as target organ-selective ER agonists for the treatment of osteoporosis and other estrogenopathies. This realization has provided the impetus for research in this area, the progress of which is discussed in this review.

Animals↗