Identification of natural ligands of retinoic acid receptor-related orphan receptor α ligand-binding domain expressed in Sf9 cells––a mass spectrometry approach

doi:10.1016/j.ab.2003.08.029

Analytical Biochemistry

Volume 323, Issue 1, 1 December 2003, Pages 139-149

https://doi.org/10.1016/j.ab.2003.08.029 Get rights and content

Abstract

The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORα-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization–mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography–mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D₃; 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORα-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.

Section snippets

Chemicals

The HPLC-grade acetonitrile and water were from Merck (Darmstadt, Germany). Cholesterol sulfate, 25-hydroxycholesterol, 7-dehydrocholesterol, horse heart myoglobin, hen egg lysozyme, and ammonium acetate were obtained from Sigma. Coactivator peptide GRIP1P (sequence GTSLKEKHKILHRLLQDSSS; MW 2277.6 Da) was synthesized by Neosystem (Strasbourg, France). N,O-BSTFA was from Pierce (Rockford, IL).

Sample preparation

The expression in Sf9 cells and purification of (His)₆RORα-LBD_271-523 and (His)₆RORα-LBD_236-523 were

Expression and purification of RORα-LBD

Two constructs of human RORα-LBD based on homology analysis were prepared. A shorter one corresponding to residues 271–523 with a cleavable N-terminal His tag (numbering according to splice variant 1 of SWISS-PROT entry P35398) had already been crystallized and analyzed by X-ray [20]. The longer LBD (residues 236–523) contained an additional 36 residues of the hinge region upstream of the LBD domain flanked by a noncleavable His tag. Production of both RORα-LBD constructs in the baculovirus

Discussion

This paper describes the purification and the analysis of (His)₆RORα-LBD by mass spectrometry under conditions allowing the detection of noncovalent complexes. HPLC/ESI-MS analysis of the purified (His)₆RORα-LBD_271-523 and (His)₆RORα-LBD_236-523 yielded average molecular masses of 31,515.5 and 34,410.2 Da, respectively, which were in agreement with those of N-terminally acetylated (His)₆RORα-LBD sequences. ESI-MS analysis performed under native conditions allowed the assessment of the presence of

Acknowledgements

We thank R. Schmitz, M. Geiser, and Y. Pouliquen for cloning, expression, and fermentation. We thank M. Blommers for NMR spectroscopy. We thank A. Berner and P. Graff for excellent technical assistance. We thank R. Amstutz, H.P. Kocher, H. Widmer, and M. Missbach for their interest and support.

References (42)

A.M. Jetten et al.
The ROR nuclear orphan receptor subfamily: critical regulators of multiple biological processes
Prog. Nucleic Acid Res.
(2001)
N. Vu-Dac et al.
Transcriptional regulation of apolipoprotein A-I gene expression by the nuclear receptor RORα
J. Biol. Chem.
(1997)
Ph. Delerive et al.
Identification of Reverα as a Novel RORα Target Gene
J. Biol. Chem.
(2002)
I. Dussault et al.
Orphan nuclear receptor RORα-deficient mice display the cerebellar defects of staggerer
Mech. Dev.
(1998)
T. Matsui et al.
An orphan nuclear receptor, mROR alpha, and its spatial expression in adult mouse brain
Mol. Brain Res.
(1995)
L.E. Morrison
Time-resolved detection of energy transfer: theory and application to immunoassay
Anal. Biochem.
(1988)
J.F. Glickman et al.
A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors
J. Biomol. Screen.
(2002)
W. Bourguet et al.
Crystal structure of a heterodimeric complex of RAR and RXR ligand-binding domains
Mol. Cell
(2000)
W. Bourguet et al.
Heterodimeric complex of RAR and RXR nuclear receptor ligand-binding domains: purification, crystallization, and preliminary X-ray diffraction analysis
Protein Expression Purific.
(2000)
I.M.L. Billas et al.
Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects
J. Biol. Chem.
(2001)

S. Dhe-Paganon et al.

Crystal structure of the HNF4α ligand binding domain in complex with endogenous fatty acid ligand

J. Biol. Chem.

(2002)

G.B. Wisely et al.

Hepatocyte Nuclear Factor 4 is a transcription factor that constitutively binds fatty acids

Structure

(2002)

T.D. Veenstra

Electrospray ionization mass spectrometry in the study of biomolecular non-covalent interactions

Biophys. Chem.

(1999)

S. Rocchi et al.

A unique PPARgamma ligand with potent insulin-sensitizing yet weak adipogenic activity

Mol. Cell

(2001)

H. Greschik et al.

Structural and functional evidence for ligand-independent transcriptional activation by the estrogen-related receptor 3

Mol. Cell

(2002)

H. Rogniaux et al.

Binding of aldose reductase inhibitors: correlation of crystallographic and mass spectrometric studies

J. Am. Soc. Mass Spectrom.

(1999)

N.A. Nwokoro et al.

Genetic disorders of cholesterol biosynthesis in mice and humans

Mol. Genet. Metabol.

(2001)

A. Chawla et al.

Nuclear receptors and lipid physiology: opening the X-files

Science

(2001)

C. Carlberg et al.

RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers

Mol. Endocrinol.

(1994)

V. Giguere et al.

Isoform-specific amino-terminal domains dictate DNA-binding properties of RORα, a novel family of orphan hormone nuclear receptors

Genes Dev.

(1994)

E. Raspé et al.

Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORα

J. Biol. Chem.

(2001)

Cited by (64)

Mechanisms of vitamin A metabolism and deficiency in the mammalian and fly visual system
2021, Developmental Biology
Vitamin A deficiency can cause human pathologies that range from blindness to embryonic malformations. This diversity is due to the lack of two major vitamin A metabolites with very different functions: the chromophore 11-cis-retinal (vitamin A aldehyde) is a critical component of the visual pigment that mediates phototransduction, while the signaling molecule all-trans-retinoic acid regulates the development of various tissues and is required for the function of the immune system.
Since animals cannot synthesize vitamin A de novo, they must obtain it either as preformed vitamin A from animal products or as carotenoid precursors from plant sources. Due to its essential role in the visual system, acute vitamin A deprivation impairs photoreceptor function and causes night blindness (poor vision under dim light conditions), while chronic deprivation results in retinal dystrophies and photoreceptor cell death. Chronic vitamin A deficiency is the leading cause of preventable childhood blindness according to the World Health Organization. Due to the requirement of vitamin A for retinoic acid signaling in development and in the immune system, vitamin A deficiency also causes increased mortality in children and pregnant women in developing countries.
Drosophila melanogaster is an excellent model to study the effects of vitamin A deprivation on the eye because vitamin A is not essential for Drosophila development and chronic deficiency does not cause lethality. Moreover, genetic screens in Drosophila have identified evolutionarily conserved factors that mediate the production of vitamin A and its cellular uptake. Here, we review our current knowledge about the role of vitamin A in the visual system of mammals and Drosophila melanogaster. We compare the molecular mechanisms that mediate the uptake of dietary vitamin A precursors and the metabolism of vitamin A, as well as the consequences of vitamin A deficiency for the structure and function of the eye.
25-hydroxycholesterol impairs neuronal and muscular development in zebrafish
2019, NeuroToxicology
Oxysterols have essential effects on brain homeostasis and their levels are often altered in neurodegenerative and neuroinflammatory diseases. Several studies have demonstrated the cytotoxic effects of 25-HC on different cell lines, however, not much is known about its effects on neurons in vivo. In this study, we examined the effects of 25-HC exposure on the nervous system development in the zebrafish. We showed that survival rate of zebrafish embryos/larvae is significantly decreased at doses of 25-HC above 40 μM. 25-HC was found to affect the motility of zebrafish larvae, primary motor axon and muscle morphology. Furthermore, larvae treated with 25-HC showed a reduced neuronal network and number of HuC-positive cells in the brain. An increased cell death was also observed in both the brain and spinal cord of zebrafish treated with 25-HC. Interestingly, administration of 25-HC at later stages of development (24 and 48 h post fertilization) had no detrimental effects on motor axons. Altogether, our findings show that elevated levels of 25-HC may have important consequences on neuronal development and cell survival.
Rethinking Nuclear Receptors as Potential Therapeutic Targets for Retinal Diseases
2016, Journal of Biomolecular Screening
Citation Excerpt :
Another study from the same group showed that GSK0060 stabilized tight junctions in human retinal microvascular endothelial cells treated with VEGF, concomitant with reducing VEGFR1/2 expression, suggesting a role in vascular permeability,82 and providing further support for pharmacologic inhibition of PPARβ/δ as the basis for therapeutic targeting of retinal neovascularization. The retinoic acid receptor-related orphan receptor alpha (RORα; NR1F1) is a lipid-sensing NR that has been shown to regulate the inflammatory response in allergy and autoimmune diseases.83-85 A recent report by Sun et al. demonstrated that the absence of RORα inhibited pathological angiogenesis in a model of oxygen-induced retinopathy, by directly impacting the expression of suppressor of cytokine signaling 3 (SOCS3), which is a critical regulator of tissue inflammation.26
Collectively, retinal diseases, including age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy, result in severe vision impairment worldwide. The absence and/or limited availability of successful drug therapies for these blinding disorders necessitates further understanding their pathobiology and identifying new targetable signaling pathways. Nuclear receptors are transcription regulators of many key aspects of human physiology, as well as pathophysiology, with reported roles in development, aging, and disease. Some of the pathways regulated by nuclear receptors include, but are not limited to, angiogenesis, inflammation, and lipid metabolic dysregulation, mechanisms also important in the initiation and development of several retinal diseases. Herein, we present an overview of the biology of three diseases affecting the posterior eye, summarize a growing body of evidence that suggests direct or indirect involvement of nuclear receptors in disease progression, and discuss the therapeutic potential of targeting nuclear receptors for treatment.
加齢性黄斑変性症、網膜色素変性症、糖尿病性網膜症などの網膜疾患は、世界中で重篤な視力障害をもたらしている。これらの失明を伴う障害に奏効する薬物療法がない、またはあっても限られていることにより、その病理生物学的理解を深め、新しい標的を設定できるシグナル伝達経路を特定することが必要になる。核内受容体はヒトの生理および病態生理における数多くの重要な側面の転写調節因子であり、成長、加齢および疾患に果たす役割が報告されている。核内受容体により調節される経路は、他にもあるが血管形成、炎症、脂質代謝調節異常などであり、幾つかの網膜疾患の発病と進展においても重要な機構である。このことから、後眼部を冒す3疾患の生態の概要を示し、疾患の進行における核内受容体の直接的または間接的関与を示唆するエビデンスに関して増えている文献を要約し、治療目的で核内受容体を標的とすることの治療可能性を考察する。
종합적으로 나이에 따른 시력 감퇴, 색소성 망막염, 당뇨성 망막증을 포함한 망막 질환은 전세계적으로 심각한 시력 손상을 야기합니다. 이러한 시력 장애에 대한 성공적인 약물 치료의 부재 및/또는 제한된 가용성으로 인해 병리 생물학에 대한 깊은 이해와 새로운 타겟팅 신호 전달 경로의 식별이 필요합니다. 핵 수용체는 병태 생리학은 물론, 인간의 생리 기능의 많은 주요 측면의 전사 조절자로, 발달, 노화 및 질병에서의 역할이 보고되었습니다. 핵 수용체에 의해 조절되는 경로 중 일부는 포함하지만 혈관 생성, 염증 및 지질 대사 조절 이상, 여러 망막 질환의 발현과 발달에 중요한 메커니즘도 포함되며 이에 국한되지 않습니다. 여기에서 우리는 후방 눈에 영향을 미치는 세 가지 질병의 생물학적 개요를 제시하고, 질환의 진행 과정에서 핵 수용체의 직접 또는 간접적 연관성을 제안하는 다양한 증거를 요약하고, 치료를 위해 핵 수용체를 표적으로 하는 치료법의 가능성에 대해 설명합니다.
在世界各地，各种视网膜疾病（包括年龄相关黄斑变性、色素性视网膜炎和糖尿病视网膜病变）共同导致严重的视力障碍。由于不存在针对这些致盲性疾病的有效药物疗法和/或疗法有限，我们有必要进一步了解这些疾病的病理，并确定新的可靶向信号通路。核受体是人类生理以及病理生理机制许多重要方面的转录调节因子，其在发育、衰老和疾病过程中的作用也不乏报道。由核受体调节的通路包括但不限于：血管生成、炎症和脂质代谢失调，其作用机制在多种视网膜疾病的发生和发展过程中也十分重要。在本文中，我们概述了影响眼后段的三种疾病的生理学机制，总结了核受体直接或间接参与疾病进展的大量证据，并论述了将核受体作为治疗靶点的治疗潜力。
在世界各地，各種視網膜疾病（包括年齡相關黃斑變性、色素性視網膜炎和糖尿病視網膜病變）共同導致嚴重的視力障礙。由於不存在針對這些致盲性疾病的有效藥物療法和/或療法有限，我們有必要進一步瞭解這些疾病的病理，並確定新的可靶向訊號通路。核受體是人類生理以及病理生理機制許多重要方面的轉錄調節因數，其在發育、衰老和疾病過程中的作用也不乏報導。由核受體調節的通路包括但不限於：血管生成、炎症和脂質代謝失調，其作用機制在多種視網膜疾病的發生和發展過程中也十分重要。在本文中，我們概述了影響眼後段的三種疾病的生理學機制，總結了核受體直接或間接參與疾病進展的大量證據，並論述了將核受體作為治療靶點的治療潛力。
Oxysterols: From cholesterol metabolites to key mediators
2016, Progress in Lipid Research
Oxysterols are cholesterol metabolites that can be produced through enzymatic or radical processes. They constitute a large family of lipids (i.e. the oxysterome) involved in a plethora of physiological processes. They can act through GPCR (e.g. EBI2, SMO, CXCR2), nuclear receptors (LXR, ROR, ERα) and through transporters or regulatory proteins. Their physiological effects encompass cholesterol, lipid and glucose homeostasis. Additionally, they were shown to be involved in other processes such as immune regulatory functions and brain homeostasis. First studied as precursors of bile acids, they quickly emerged as interesting lipid mediators. Their levels are greatly altered in several pathologies and some oxysterols (e.g. 4β-hydroxycholesterol or 7α-hydroxycholestenone) are used as biomarkers of specific pathologies. In this review, we discuss the complex metabolism and molecular targets (including binding properties) of these bioactive lipids in human and mice. We also discuss the genetic mouse models currently available to interrogate their effects in pathophysiological settings. We also summarize the levels of oxysterols reported in two key organs in oxysterol metabolism (liver and brain), plasma and cerebrospinal fluid. Finally, we consider future opportunities and directions in the oxysterol field in order to gain a better insight and understanding of the complex oxysterol system.
Retinoid-related orphan receptor alpha and the regulation of lipid homeostasis
2012, Journal of Steroid Biochemistry and Molecular Biology
Many nuclear hormone receptors (NRs) control lipid, glucose and energy homeostasis in an organ specific manner. Concordantly, dysfunctional NR signalling results in metabolic disease. The Retinoic acid receptor-related orphan receptor alpha (RORα), a member of the NR1F subgroup, is expressed in metabolic tissues. Previous studies identified the role of this NR in dyslipidemia, apo-lipoprotein metabolism and atherosclerosis. Recent data is underscoring the significant role of this orphan NR in the regulation of phase I/II metabolism (bile acids, xenobiotics, steroids etc.), adiposity, insulin signalling, and glucose tolerance. Moreover, oxygenated sterols, have been demonstrated to function as native ligands and inverse agonists. This review focuses on the rapidly emerging and evolving role of RORα in the control of lipid and glucose homeostasis in major mass metabolic tissues.
Article from the special issue orphan receptors.
Melatonin membrane receptors in peripheral tissues: Distribution and functions
2012, Molecular and Cellular Endocrinology
Many of melatonin’s actions are mediated through interaction with the G-protein coupled membrane bound melatonin receptors type 1 and type 2 (MT1 and MT2, respectively) or, indirectly with nuclear orphan receptors from the RORα/RZR family. Melatonin also binds to the quinone reductase II enzyme, previously defined the MT3 receptor. Melatonin receptors are widely distributed in the body; herein we summarize their expression and actions in non-neural tissues. Several controversies still exist regarding, for example, whether melatonin binds the RORα/RZR family. Studies of the peripheral distribution of melatonin receptors are important since they are attractive targets for immunomodulation, regulation of endocrine, reproductive and cardiovascular functions, modulation of skin pigmentation, hair growth, cancerogenesis, and aging. Melatonin receptor agonists and antagonists have an exciting future since they could define multiple mechanisms by which melatonin modulates the complexity of such a wide variety of physiological and pathological processes.

View all citing articles on Scopus

View full text

Identification of natural ligands of retinoic acid receptor-related orphan receptor α ligand-binding domain expressed in Sf9 cells––a mass spectrometry approach

Abstract

Section snippets

Chemicals

Sample preparation

Expression and purification of RORα-LBD

Discussion

Acknowledgements

Prog. Nucleic Acid Res.

J. Biol. Chem.

J. Biol. Chem.

Mech. Dev.

Mol. Brain Res.

Anal. Biochem.

J. Biomol. Screen.

Mol. Cell

Protein Expression Purific.

J. Biol. Chem.

J. Biol. Chem.

Structure

Biophys. Chem.

Mol. Cell

Mol. Cell

J. Am. Soc. Mass Spectrom.

Mol. Genet. Metabol.

Nuclear receptors and lipid physiology: opening the X-files

Science

RZRs, a new family of retinoid-related orphan receptors that function as both monomers and homodimers

Mol. Endocrinol.

Isoform-specific amino-terminal domains dictate DNA-binding properties of RORα, a novel family of orphan hormone nuclear receptors

Genes Dev.

Transcriptional regulation of apolipoprotein C-III gene expression by the orphan nuclear receptor RORα

J. Biol. Chem.