Peter J. Kushner

Bio: Peter J. Kushner is an academic researcher from University of California, San Francisco. The author has contributed to research in topics: Estrogen receptor & Estrogen receptor alpha. The author has an hindex of 45, co-authored 60 publications receiving 14669 citations. Previous affiliations of Peter J. Kushner include University of Illinois at Chicago & Baxter International.

DIFFERENTIAL LIGAND ACTIVATION OF ESTROGEN RECEPTORS ER$g(a) AND ER$g(b) AT AP1 SITES

Abstract: This invention provides methods of screening test compounds for the ability to activate or inhibit estrogen receptor beta (ER beta ) mediated gene activation at an AP1 site. In particular, the methods involve providing a cell comprising an estrogen receptor beta (ER beta ), AP1 proteins, and a construct comprising a promoter comprising an AP1 site which regulates expression of a first reporter gene. The cell is contacted with the test compound and changes in expression levels of the reporter gene are detected indicating whether the test compounds activate transcription, inactivate transcription or have no effect at the AP1 site.

Structure and specificity of nuclear receptor-coactivator interactions.

Abstract: Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing α-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic α-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.

Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor.

Abstract: A conserved region in the hormone-dependent activation domain AF2 of nuclear receptors plays an important role in transcriptional activation. We have characterized a novel nuclear protein, RIP140, that specifically interacts in vitro with this domain of the estrogen receptor. This interaction was increased by estrogen, but not by anti-estrogens and the in vitro binding capacity of mutant receptors correlates with their ability to stimulate transcription. RIP140 also interacts with estrogen receptor in intact cells and modulates its transcriptional activity in the presence of estrogen, but not the anti-estrogen 4-hydroxytamoxifen. In view of its widespread expression in mammalian cells, RIP140 may interact with other members of the superfamily of nuclear receptors and thereby act as a potential co-activator of hormone-regulated gene transcription.

Cloning of a novel receptor expressed in rat prostate and ovary.

Abstract: We have cloned a novel member of the nuclear receptor superfamily. The cDNA of clone 29 was isolated from a rat prostate cDNA library and it encodes a protein of 485 amino acid residues with a calculated molecular weight of 54.2 kDa. Clone 29 protein is unique in that it is highly homologous to the rat estrogen receptor (ER) protein, particularly in the DNA-binding domain (95%) and in the C-terminal ligand-binding domain (55%). Expression of clone 29 in rat tissues was investigated by in situ hybridization and prominent expression was found in prostate and ovary. In the prostate clone 29 is expressed in the epithelial cells of the secretory alveoli, whereas in the ovary the granuloma cells in primary, secondary, and mature follicles showed expression of clone 29. Saturation ligand-binding analysis of in vitro synthesized clone 29 protein revealed a single binding component for 17beta-estradiol (E2) with high affinity (Kd= 0.6 nM). In ligand-competition experiments the binding affinity decreased in the order E2 > diethylstilbestrol > estriol > estrone > 5alpha-androstane-3beta,17beta-diol >> testosterone = progesterone = corticosterone = 5alpha-androstane-3alpha,17beta-diol. In cotransfection experiments of Chinese hamster ovary cells with a clone 29 expression vector and an estrogen-regulated reporter gene, maximal stimulation (about 3-fold) of reporter gene activity was found during incubation with 10 nM of E2. Neither progesterone, testosterone, dexamethasone, thyroid hormone, all-trans-retinoic acid, nor 5alpha-androstane-3alpha,I7beta-diol could stimulate reporter gene activity, whereas estrone and 5alpha-androstane-3beta,17beta-diol did. We conclude that clone 29 cDNA encodes a novel rat ER, which we suggest be named rat ERbeta to distinguish it from the previously cloned ER (ERalpha) from rat uterus.

Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta

Abstract: The rat estrogen receptor (ER) exists as two subtypes, ER alpha and ER beta, which differ in the C-terminal ligand binding domain and in the N-terminal transactivation domain. In this study we investigated the messenger RNA expression of both ER subtypes in rat tissues by RT-PCR and compared the ligand binding specificity of the ER subtypes. Saturation ligand binding analysis of in vitro synthesized human ER alpha and rat ER beta protein revealed a single binding component for 16 alpha-iodo-17 beta-estradiol with high affinity [dissociation constant (Kd) = 0.1 nM for ER alpha protein and 0.4 nM for ER beta protein]. Most estrogenic substances or estrogenic antagonists compete with 16 alpha-[125I]iodo-17 beta-estradiol for binding to both ER subtypes in a very similar preference and degree; that is, diethylstilbestrol > hexestrol > dienestrol > 4-OH-tamoxifen > 17 beta-estradiol > coumestrol, ICI-164384 > estrone, 17 alpha-estradiol > nafoxidine, moxestrol > clomifene > estriol, 4-OH-estradiol > tamoxifen, 2-OH-estradiol, 5-androstene-3 beta, 17 beta-diol, genistein for the ER alpha protein and dienestrol > 4-OH-tamoxifen > diethylstilbestrol > hexestrol > coumestrol, ICI-164384 > 17 beta-estradiol > estrone, genistein > estriol > nafoxidine, 5-androstene-3 beta, 17 beta-diol > 17 alpha-estradiol, clomifene, 2-OH-estradiol > 4-OH-estradiol, tamoxifen, moxestrol for the ER beta protein. The rat tissue distribution and/or the relative level of ER alpha and ER beta expression seems to be quite different, i.e. moderate to high expression in uterus, testis, pituitary, ovary, kidney, epididymis, and adrenal for ER alpha and prostate, ovary, lung, bladder, brain, uterus, and testis for ER beta. The described differences between the ER subtypes in relative ligand binding affinity and tissue distribution could contribute to the selective action of ER agonists and antagonists in different tissues.

Autonomic healing of polymer composites

Abstract: Structural polymers are susceptible to damage in the form of cracks, which form deep within the structure where detection is difficult and repair is almost impossible. Cracking leads to mechanical degradation of fibre-reinforced polymer composites; in microelectronic polymeric components it can also lead to electrical failure. Microcracking induced by thermal and mechanical fatigue is also a long-standing problem in polymer adhesives. Regardless of the application, once cracks have formed within polymeric materials, the integrity of the structure is significantly compromised. Experiments exploring the concept of self-repair have been previously reported, but the only successful crack-healing methods that have been reported so far require some form of manual intervention. Here we report a structural polymeric material with the ability to autonomically heal cracks. The material incorporates a microencapsulated healing agent that is released upon crack intrusion. Polymerization of the healing agent is then triggered by contact with an embedded catalyst, bonding the crack faces. Our fracture experiments yield as much as 75% recovery in toughness, and we expect that our approach will be applicable to other brittle materials systems (including ceramics and glasses).

Intrinsically unstructured proteins and their functions.

Abstract: Many gene sequences in eukaryotic genomes encode entire proteins or large segments of proteins that lack a well-structured three-dimensional fold. Disordered regions can be highly conserved between species in both composition and sequence and, contrary to the traditional view that protein function equates with a stable three-dimensional structure, disordered regions are often functional, in ways that we are only beginning to discover. Many disordered segments fold on binding to their biological targets (coupled folding and binding), whereas others constitute flexible linkers that have a role in the assembly of macromolecular arrays.