NIH grant: Surveying type I nuclear receptor activation by environmental toxins (1R21ES038311-01A1)

retained text

NIH grant 1R21ES038311-01A1: Surveying type I nuclear receptor activation by environmental toxins
Institute: {'code': 'ES', 'abbreviation': 'NIEHS', 'name': 'National Institute of Environmental Health Sciences', 'admin_org_id': '12500022', 'admin_funding_url': ''}. PI: Justin Matthew Holub. Award: $404163.
Start: 2026-08-07T00:00:00.
PROJECT SUMMARY
Endocrine-disrupting chemicals (EDCs) and nanoplastics (NPs) are environmental toxins that interfere with the
normal function of the endocrine system by binding to type I nuclear receptors (NR), a family of ligand-mediated
transcription factors that act as key regulators of metabolism, development, reproduction, and cell proliferation.
Members of the type I NR family, including estrogen receptors (ER), androgen receptors (AR), and progesterone
receptors (PR), are activated through the binding of endogenous steroid hormones and exert their biological
effects by controlling the expression of steroid-responsive genes in the nucleus. Unfortunately, many EDCs and
NPs compete for ligand-binding domains (LBDs) of type I NRs by mimicking the size and structure of natural
steroid hormones. If left untreated, such aberrant interactions often lead to debilitating physiological disorders,
including neurodegeneration, inflammation, autoimmunity and cancer. Broadly speaking, the LBDs of type I NRs
undergo moderate structural changes upon ligand binding that involves transition of a dynamic C-terminal helix
(H12) from an extended (inactive) state to a well-folded (active) state. Our laboratory has developed a novel, in
vitro assay that uses a convenient fluorescence-based readout to survey ligand-mediated H12 transitions within
the ER-LBD. The assay, termed ‘FlAsH-ER’, employs bipartite tetracysteine display coupled with the biarsenical
profluorophore FlAsH-EDT2 to monitor H12 transitions as a function of fluorescence intensity. Using FlAsH-ER,
we have successfully monitored ligand-dependent structural changes of H12 in ER-LBDs bound to various
estrogenic ligands and selective estrogen receptor modulators, and tracked ligand-independent H12 transitions
in constitutively active ER mutants expressed in endocrine-resistant breast cancers. We now seek to utilize the
FlAsH-ER assay to directly study how EDCs and NPs affect ligand-mediated H12 transitions within type I NRs.
In Specific Aim 1 of this proposal, our work will focus on applying the FlAsH-ER assay to monitor H12 transitions
within ERs that have been exposed to EDCs or NPs. This will be accomplished by treating wild-type or mutant
ERs with discrete EDCs or NPs at concentrations that reflect real-world exposure levels and monitoring H12
transitions as they relate to ligand binding. Notably, such studies will allow us to directly determine how EDCs
and NPs affect active-state (or inactive-state) H12 transitions within the ER. In Specific Aim 2, we expand the
utility of the FlAsH-ER assay by commuting the technique to other type I NR LBDs. Here, we will use site-directed
mutagenesis to generate tetracysteine-tagged AR-LBDs and PR-LBDs that will recapitulate an optimized FlAsH-
binding motif upon interaction with ligand. Correlative ‘FlAsH-AR’ and ‘FlAsH-PR’ assays will then be used to
determine how endogenous steroids and environmental toxins, including EPCs and NPs, affect active-state H12
transitions within each receptor subtype. Collectively, results obtained from the work outlined in this proposal will
determine the molecular mechanisms that drive ligand-mediated activation of type I NRs and will facilitate the
development of lead compounds to treat endocrine disorders that arise from exposure to environmental toxins.

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