scholarly journals HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

2019 ◽  
Author(s):  
Timothy S. Strutzenberg ◽  
Ruben Garcia-Ordonez ◽  
Scott Novick ◽  
HaJeung Park ◽  
Mi Ra Chang ◽  
...  
Keyword(s):  
Nuclear Receptor ◽  
Receptor Activation ◽  
Site Directed Mutagenesis ◽  
Orphan Nuclear Receptor ◽  
Chemical Probes ◽  
Structural Determinants ◽  
Synthetic Ligand ◽  
Hdx Ms

ABSTRACTMembers of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism1 to inflammation2 and cancer3. As ligand-gated transcription factors, synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology4. However, nearly half of NRs do not have specific cognate ligands or its unclear if they possess ligand dependent activities and these receptors are called orphans. Here we demonstrate that ligand-dependent action of the orphan nuclear receptor RORγ can be defined by selectively disrupting putative endogenous—but not synthetic—ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings are corroborated with X-ray co-crystallography and site-directed mutagenesis to collectively reveal the structural determinants of RORγ ligand-dependent activation, critical for designing full agonists for application in cancer immunotherapy. Combined these observations support a model of receptor activation to more accurately describe RORγ pharmacology. Likewise, this ‘bump-and-hole’ inspired approach could be extended to other orphan NRs to explore the ligand-dependent activities that are important for defining pharmacology.

scholarly journals HDX-MS reveals structural determinants for RORγ hyperactivation by synthetic agonists

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Timothy S Strutzenberg ◽  
Ruben D Garcia-Ordonez ◽  
Scott J Novick ◽  
HaJeung Park ◽  
Mi Ra Chang ◽  
...  
Keyword(s):  
Small Molecules ◽  
Structural Dynamics ◽  
Site Directed Mutagenesis ◽  
Chemical Probes ◽  
Structural Determinants ◽  
X Ray ◽  
Synthetic Ligand ◽  
Hdx Ms

Members of the nuclear receptor (NR) superfamily regulate both physiological and pathophysiological processes ranging from development and metabolism to inflammation and cancer. Synthetic small molecules targeting NRs are often deployed as therapeutics to correct aberrant NR signaling or as chemical probes to explore the role of the receptor in physiology. Nearly half of NRs do not have specific cognate ligands (termed orphan NRs) and it’s unclear if they possess ligand dependent activities. Here we demonstrate that ligand-dependent action of the orphan RORγ can be defined by selectively disrupting putative endogenous—but not synthetic—ligand binding. Furthermore, the characterization of a library of RORγ modulators reveals that structural dynamics of the receptor assessed by HDX-MS correlate with activity in biochemical and cell-based assays. These findings, corroborated with X-ray co-crystallography and site-directed mutagenesis, collectively reveal the structural determinants of RORγ activation, which is critical for designing RORγ agonists for cancer immunotherapy.

scholarly journals Characterization of the Structural Determinants of the Ubiquitin-Dependent Proteasomal Degradation of Human Hepatic Tryptophan 2,3-Dioxygenase

2021 ◽  
Author(s):  
Yi Liu ◽  
Sung Mi Kim ◽  
YongQiang Wang ◽  
Shay Karkashon ◽  
Ariel Lewis-Ballester ◽  
...  
Keyword(s):  
Proteasomal Degradation ◽  
Site Directed Mutagenesis ◽  
Liver Protein ◽  
Lysosomal Degradation ◽  
Structural Determinants ◽  
The Poor ◽  
Structural Relevance ◽  
Positively Charged

Human hepatic tryptophan 2,3-dioxygenase (hTDO) is a homotetrameric hemoprotein. It is one of the most rapidly degraded liver proteins with a half-life (t1/2) of ~2.3 h, relative to an average t1/2 of ~2–3 days for total liver protein. The molecular mechanism underlying the poor longevity of hTDO remains elusive. Previously, we showed that hTDO could be recognized and ubiquitinated by two E3 ubiquitin (Ub) ligases, gp78/AMFR and CHIP, and subsequently degraded via Ub-dependent proteasomal degradation (UPD) pathway. Additionally, we identified 15 ubiquitination K sites and demonstrated that Trp-binding to an exosite impeded its proteolytic degradation. Here we further established autophagic lysosomal degradation (ALD) as an alternative back-up pathway for cellular hTDO degradation. In addition, with protein kinases A and C, we identified 13 phosphorylated Ser/Thr (pS/pT) sites. Mapping these pS/pT sites on the hTDO surface revealed their propinquity to acidic Asp/Glu (D/E) residues engendering negatively charged DEpSpT clusters vicinal to the ubiquitination K sites over the entire protein surface. Through site-directed mutagenesis of positively charged patches of gp78, previously documented to interact with the DEpSpT clusters in other target proteins, we uncovered the likely role of the DEpSpT clusters in the molecular recognition of hTDO by gp78 and plausibly other E3 Ub-ligases. Furthermore, cycloheximide-chase analyses revealed the critical structural relevance of the disordered N- and C-termini not only in the Ub-ligase recognition, but also in the proteasome engagement. Together, the surface DEpSpT clusters and the N- and C-termini constitute an intrinsic bipartite degron for hTDO physiological turnover.

scholarly journals Molecular cloning and characterization of chicken orphan nuclear receptor cTR21

2003 ◽  
Vol 132 (3) ◽  
pp. 474-484 ◽  
Author(s):  
Sabyasachi Sanyal ◽  
Chirstoph Handschin ◽  
Michael Podvinec ◽  
Kwang-Hoon Song ◽  
Han-Jong Kim ◽  
...  
Keyword(s):  
Molecular Cloning ◽  
Nuclear Receptor ◽  
Orphan Nuclear Receptor

scholarly journals Characterization of the Nurr1 ligand-binding domain co-activator interaction surface

2006 ◽  
Vol 37 (2) ◽  
pp. 317-326 ◽  
Author(s):  
Nikolaos Volakakis ◽  
Michal Malewicz ◽  
Banafsheh Kadkhodai ◽  
Thomas Perlmann ◽  
Gerard Benoit
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Close Association ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
Site Directed Mutagenesis ◽  
Orphan Nuclear Receptor ◽  
Hydrophobic Region ◽  
Binding Surface

The recently solved crystal structure of the orphan nuclear receptor (NR) Nurr1 ligand-binding domain (LBD) showed that Nurr1 lacks a cavity for ligand binding and a canonical NR co-activator-binding site. Computer modeling of the Nurr1 LBD structure identified a hydrophobic region on the surface of the Nurr1 LBD that was positioned on the opposite side from the classical co-activator-binding site. Site-directed mutagenesis demonstrated that this region is critical for the activity of the Nurr1 LBD. Most mutations introduced in this region reduced or abolished transcriptional activity of the Nurr1 LBD, but mutation at lysine (K577) resulted in a drastically increased activity. Moreover, the activity of the Nurr1 LBD was shown to correlate with a propensity for proteasome-dependent degradation revealing a close association between activity and Nurr1 protein turnover. These data provide novel insights into the mechanisms of transcription via the Nurr1 LBD and identify an alternative co-activator-binding surface that is unique to the NR4A family of NRs.

scholarly journals Functional characterization of an orphan nuclear receptor, Rev-ErbAα, in chondrocytes and its potential role in osteoarthritis

2006 ◽  
Vol 54 (11) ◽  
pp. 3513-3522 ◽  
Author(s):  
P. Chaturvedi ◽  
M. Pratta ◽  
K. Steplewski ◽  
J. Connor ◽  
S. Kumar
Keyword(s):  
Nuclear Receptor ◽  
Potential Role ◽  
Functional Characterization ◽  
Orphan Nuclear Receptor
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