GST-tagged mouse estrogen receptor α-transactivation domain fusion protein is specifically degraded during its over-expression in E. coli and purification

2009 ◽  
Vol 37 (3) ◽  
pp. 1335-1340 ◽  
Author(s):  
M. K. Thakur ◽  
Swati Ghosh
Keyword(s):  
Estrogen Receptor ◽  
Fusion Protein ◽  
Estrogen Receptor Α ◽  
Transactivation Domain ◽  
Over Expression ◽  
E Coli ◽  
Domain Fusion

scholarly journals PARP7 and Mono-ADP-Ribosylation Negatively Regulate Estrogen Receptor α Signaling in Human Breast Cancer Cells

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 623
Author(s):  
Marit Rasmussen ◽  
Susanna Tan ◽  
Venkata S. Somisetty ◽  
David Hutin ◽  
Ninni Elise Olafsen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Protein Modification ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Transactivation Domain ◽  
Adp Ribosylation ◽  
Protein Levels ◽  
17Β Estradiol ◽  
Mcf 7

ADP-ribosylation is a post-translational protein modification catalyzed by a family of proteins known as poly-ADP-ribose polymerases. PARP7 (TIPARP; ARTD14) is a mono-ADP-ribosyltransferase involved in several cellular processes, including responses to hypoxia, innate immunity and regulation of nuclear receptors. Since previous studies suggested that PARP7 was regulated by 17β-estradiol, we investigated whether PARP7 regulates estrogen receptor α signaling. We confirmed the 17β-estradiol-dependent increases of PARP7 mRNA and protein levels in MCF-7 cells, and observed recruitment of estrogen receptor α to the promoter of PARP7. Overexpression of PARP7 decreased ligand-dependent estrogen receptor α signaling, while treatment of PARP7 knockout MCF-7 cells with 17β-estradiol resulted in increased expression of and recruitment to estrogen receptor α target genes, in addition to increased proliferation. Co-immunoprecipitation assays revealed that PARP7 mono-ADP-ribosylated estrogen receptor α, and mass spectrometry mapped the modified peptides to the receptor’s ligand-independent transactivation domain. Co-immunoprecipitation with truncated estrogen receptor α variants identified that the hinge region of the receptor is required for PARP7-dependent mono-ADP-ribosylation. These results imply that PARP7-mediated mono-ADP-ribosylation may play an important role in estrogen receptor positive breast cancer.

scholarly journals Differential Regulation of Estrogen-Inducible Proteolysis and Transcription by the Estrogen Receptor α N Terminus

2005 ◽  
Vol 25 (13) ◽  
pp. 5417-5428 ◽  
Author(s):  
Christopher C. Valley ◽  
Raphaël Métivier ◽  
Natalia M. Solodin ◽  
Amy M. Fowler ◽  
Mara T. Mashek ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Transcriptional Activity ◽  
Target Genes ◽  
Estrogen Receptor Α ◽  
Transactivation Domain ◽  
Related Factors ◽  
Additional Element ◽  
Transactivation Domains ◽  
Ubiquitin Proteasome ◽  
Transcriptional Complex

ABSTRACT The ubiquitin-proteasome pathway has emerged as an important regulatory mechanism governing the activity of several transcription factors. While estrogen receptor α (ERα) is also subjected to rapid ubiquitin-proteasome degradation, the relationship between proteolysis and transcriptional regulation is incompletely understood. Based on studies primarily focusing on the C-terminal ligand-binding and AF-2 transactivation domains, an assembly of an active transcriptional complex has been proposed to signal ERα proteolysis that is in turn necessary for its transcriptional activity. Here, we investigated the role of other regions of ERα and identified S118 within the N-terminal AF-1 transactivation domain as an additional element for regulating estrogen-induced ubiquitination and degradation of ERα. Significantly, different S118 mutants revealed that degradation and transcriptional activity of ERα are mechanistically separable functions of ERα. We find that proteolysis of ERα correlates with the ability of ERα mutants to recruit specific ubiquitin ligases regardless of the recruitment of other transcription-related factors to endogenous model target genes. Thus, our findings indicate that the AF-1 domain performs a previously unrecognized and important role in controlling ligand-induced receptor degradation which permits the uncoupling of estrogen-regulated ERα proteolysis and transcription.

scholarly journals Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth

2010 ◽  
Vol 128 (2) ◽  
pp. 357-368 ◽  
Author(s):  
Robert S. Tolhurst ◽  
Ross S. Thomas ◽  
Fiona J. Kyle ◽  
Hetal Patel ◽  
Manikandan Periyasamy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Estrogen Receptor ◽  
Cell Survival ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Estrogen Receptor Α ◽  
Over Expression

Crystallization of an intact GST-estrogen receptor hormone binding domain fusion protein

1998 ◽  
Vol 54 (3) ◽  
pp. 423-426 ◽  
Author(s):  
John M. Lally ◽  
Richard H. Newman ◽  
Philip P. Knowles ◽  
Suhail Islam ◽  
Arnold I. Coffer ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
Fusion Protein ◽  
Binding Domain ◽  
Thin Plates ◽  
Hormone Binding ◽  
X Ray Diffraction ◽  
Visible Image ◽  
Domain Fusion ◽  
Hormone Binding Domain ◽  
Gst Fusion Proteins

Crystals of an intact GST–estrogen receptor hormone binding domain fusion protein have been grown from solutions of MPD. The crystals grew as clusters of thin plates and needles of maximum dimensions 100 × 20 × 1 µm but were unsuitable for X-ray diffraction analysis. However, examination by electron microscopy shows an ordered lattice in which the protein molecules are clearly visible. Image analysis of electron micrographs of the protein crystals revealed electron stain-excluding density which showed a two-domain trimeric structure in projection, with each molecule of dimensions 12.0 × 5.0 nm diameter. The use of GST-fusion proteins in crystallization are discussed.

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