Quantitative Nanoproteomics for Protein Complexes (QNanoPX) Related to Estrogen Transcriptional Action

Abstract

We developed an integrated proteomics approach using a chemically functionalized gold nanoparticle (AuNP) as a novel probe for affinity purification to analyze a large protein complex in vivo. We then applied this approach to globally map the transcriptional activation complex of the estrogen response element (ERE). This approach was designated as quantitative nanoproteomics for protein complexes (QNanoPX). In this approach, the positive AuNP-ERE probes were functionalized with polyethylene glycol (PEG), and the consensus sequence of ERE and negative AuNP-PEG probes were functionalized with PEG without the ERE via a thiolated self-assembly monolayer technique. The AuNP-ERE probe had substantially low nonspecific binding and high solubility, which resulted in a 20-fold enrichment of the factor compared with gel beads. In addition, the surface-only binding allows the probe to capture a large protein complex without any restrictions due to pore size. The affinity purification method was combined with MS-based quantitative proteomics and statistical methods to reveal the components of the ERE complex in MCF-7 cells and to identify those components within the complex that were altered by the presence of 17␤-estradiol (E2).

Identification of a Hormone-regulated Dynamic Nuclear Actin Network Associated with Estrogen Receptor α in Human Breast Cancer Cell Nuclei

“…It is worth mentioning that Arp2/3 and myosin 1c have been described to have a role with ␤-actin not only in regulating RNA polymerase II activity (35,48,49) but also in long range chromatin interactions involving hormoneresponsive genes and mediated by ER␣ (9). A recent study identified, by quantitative nanoproteomics (QNanoPX), several ERE-binding proteins from MCF-7 cell nuclei, including most of the ER␣ interactors reported in Table II (50). In vitro binding to ERE of a significant number of these proteins, including ER␣ itself, was found to be increased by estrogen treatment of the cells, suggesting that they might belong to multiprotein complexes also comprising the receptor.…”

Enrichment and Detection of Rare Proteins with Aptamer-Conjugated Gold Nanorods

“…5 Since the band intensities for this nonspecifically captured α-thrombin were so low, it can be concluded that the nonspecific capture of α-thrombin in the absence of the affinity tag conjugation was negligible.…”

“…Gold nanorods were functionalized with thiol-PEG (MW 5000) to avoid agglomeration and decrease nonspecific binding. 5,27–31 …”

Estrogen Alters the Splicing of Type 1 Corticotropin-Releasing Hormone Receptor in Breast Cancer Cells

“…transcription associated with the proliferative response (35,36). We explored the possibility that estrogens can alter MCF7 cellular sensitivity to CRH by characterizing the effects of estrogens on CRH potential to induce kinase phosphorylation.…”