Influence of Domain Interactions on Conformational Mobility of the Progesterone Receptor Detected by Hydrogen/Deuterium Exchange Mass Spectrometry

Goswami, Devrishi; Callaway, Celetta; Pascal, Bruce D.; Kumar, Raj; Edwards, Dean P.; Griffin, Patrick R.

doi:10.1016/j.str.2014.04.013

Cited by 30 publications

(16 citation statements)

References 69 publications

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“…Solution-phase amide HDX was performed with a fully automated system as described previously with minor modifications 31, 32 . For differential HDX experiments, 5μL of a 10μM RORγt LBD solution (Apo or in complex with 10-excess compound) was diluted to 25μL with D 2 O-containing HDX buffer, and incubated at 4°C for; 10s, 30s, 60s, 900s, and 3,600s.…”

Section: Methodsmentioning

confidence: 99%

Synthetic RORγt Agonists Enhance Protective Immunity

et al. 2016

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The T cell specific RORγ isoform RORγt has been shown to be the key lineage-defining transcription factor to initiate the differentiation program of TH17 and Tc17 cells, cells that have demonstrated anti-tumor efficacy. RORγt controls gene networks that enhance immunity including increased IL17 production and decreased immune suppression. Both synthetic and putative endogenous agonists of RORγt have been shown to increase the basal activity of RORγt enhancing TH17 cell proliferation. Here we show that activation of RORγt using synthetic agonists drives proliferation of TH17 cells while decreasing levels of the immune checkpoint protein PD-1, a mechanism that should enhance anti-tumor immunity while blunting tumor associated adaptive immune resistance. Interestingly, putative endogenous agonists drive proliferation of TH17 cells but do not repress PD-1. These findings suggest that synthetic agonists of RORγt should activate TC17/TH17 cells (with concomitant reduction in the Tregs population), repress PD-1, and produce IL17 in situ (a factor associated with good prognosis in cancer). Enhanced immunity and blockage of immune checkpoints has transformed cancer treatment, thus such a molecule would provide a unique approach for the treatment of cancer.

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Section: Methodsmentioning

confidence: 99%

Synthetic RORγt Agonists Enhance Protective Immunity

et al. 2016

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“…PR associate with multiple proteins, including nuclear receptor coactivators, which stabilize the receptor complex and enhance transcription ( Hill et al, 2012 ; Goswami et al, 2014 ; Simons et al, 2014 ). In the female mouse hypothalamus, the PR isoforms are differentially expressed with steroid receptor coactivator-1 (SRC-1, also named NCOA1) and SRC-2 (NCOA2; Acharya et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent

Acharya

Nettles

Sellers

et al. 2017

eNeuro

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Progestins bind to the progestin receptor (PR) isoforms, PR-A and PR-B, in brain to influence development, female reproduction, anxiety, and stress. Hormone-activated PRs associate with multiple proteins to form functional complexes. In the present study, proteins from female mouse hypothalamus that associate with PR were isolated using affinity pull-down assays with glutathione S-transferase–tagged mouse PR-A and PR-B. Using complementary proteomics approaches, reverse phase protein array (RPPA) and mass spectrometry, we identified hypothalamic proteins that interact with PR in a ligand-dependent and isoform-specific manner and were confirmed by Western blot. Synaptic proteins, including synapsin-I and synapsin-II, interacted with agonist-bound PR isoforms, suggesting that both isoforms function in synaptic plasticity. In further support, synaptogyrin-III and synapsin-III associated with PR-A and PR-B, respectively. PR also interacted with kinases, including c-Src, mTOR, and MAPK1, confirming phosphorylation as an integral process in rapid effects of PR in the brain. Consistent with a role in transcriptional regulation, PR associated with transcription factors and coactivators in a ligand-specific and isoform-dependent manner. Interestingly, both PR isoforms associated with a key regulator of energy homeostasis, FoxO1, suggesting a novel role for PR in energy metabolism. Because many identified proteins in this PR interactome are synaptic proteins, we tested the hypothesis that progestins function in synaptic plasticity. Indeed, progesterone enhanced synaptic density, by increasing synapsin-I–positive synapses, in rat primary cortical neuronal cultures. This novel combination of RPPA and mass spectrometry allowed identification of PR action in synaptic remodeling and energy homeostasis and reveals unique roles for progestins in brain function and disease.

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“…Coupling this approach with modern high-resolution mass spectrometry to monitor deuterium incorporation enables precise and sensitive data collection (<1ug/experiment at low μM concentration), and interrogation of large proteins and complexes [1-3]. Differential HDX-MS analysis of a protein under different conditions (e.g apo vs. holo protein) has emerged as an important tool to probe the effects of chemical modifications, mutations, and binding events on protein stability and conformational dynamics (Figure 1).…”

Section: Hdx-ms Introductionmentioning

confidence: 99%

HDX-MS guided drug discovery: small molecules and biopharmaceuticals

Marciano

Dharmarajan

Griffin

2014

Current Opinion in Structural Biology

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Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS or DXMS) has emerged as an important tool for the development of small molecule therapeutics and biopharmaceuticals. Central to these advances have been improvements to automated HDX-MS platforms and software that allow for the rapid acquisition and processing of experimental data. Correlating the HDX-MS profile of large numbers of ligands with their functional outputs has enabled the development of structure activity relationships (SAR) and delineation of ligand classes based on functional selectivity. HDX-MS has also been applied to address many of the unique challenges posed by the continued emergence of biopharmaceuticals. Here we review the latest applications of HDX-MS to drug discovery, recent advances in technology and software, and provide perspective on future outlook.

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Influence of Domain Interactions on Conformational Mobility of the Progesterone Receptor Detected by Hydrogen/Deuterium Exchange Mass Spectrometry

Cited by 30 publications

References 69 publications

Synthetic RORγt Agonists Enhance Protective Immunity

Synthetic RORγt Agonists Enhance Protective Immunity

The Progestin Receptor Interactome in the Female Mouse Hypothalamus: Interactions with Synaptic Proteins Are Isoform Specific and Ligand Dependent

HDX-MS guided drug discovery: small molecules and biopharmaceuticals

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