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

Bitsch, Francis; Aichholz, Reiner; Kallen, Joerg; Geisse, Sabine; Fournier, Brigitte; Schlaeppi, Jean Marc

doi:10.1016/j.ab.2003.08.029

Cited by 68 publications

(57 citation statements)

References 38 publications

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“…1D) that matched a previously described spectrum for derivatized cholesterol ( Fig. 1F; Bitsch et al 2003). Based on these two independent modes of MS-ESI/MS and GC/MS-we conclude that the DHR96 LBD is capable of binding a single molecule of cholesterol.…”

Section: Dhr96 Binds Cholesterolmentioning

confidence: 48%

The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis

Horner¹,

Pardee²,

et al. 2009

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Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. Here we show that the Drosophila DHR96 nuclear receptor binds cholesterol and is required for the coordinate transcriptional response of genes that are regulated by cholesterol and involved in cholesterol uptake, trafficking, and storage. DHR96 mutants die when grown on low levels of cholesterol and accumulate excess cholesterol when maintained on a high-cholesterol diet. The cholesterol accumulation phenotype can be attributed to misregulation of npc1b, an ortholog of the mammalian Niemann-Pick C1-like 1 gene NPC1L1, which is essential for dietary cholesterol uptake. These studies define DHR96 as a central regulator of cholesterol homeostasis. Cholesterol is an essential component of cell membranes that influences the permeability and fluidity of the lipid bilayer. Cholesterol also acts as a precursor for steroid hormone biosynthesis and contributes to cell-cell signaling pathways. These critical cellular functions are supported by regulatory mechanisms that maintain normal cholesterol levels and prevent hypercholesterolemia, which is a major risk factor for cardiovascular disease in humans. Cholesterol homeostasis in vertebrates is achieved primarily through de novo synthesis and dietary uptake (Ikonen 2008). Although extensive studies have defined a central role for the sterol regulatory elementbinding protein (SREBP) family of transcription factors in controlling cholesterol synthesis (Brown and Goldstein 1997), the mechanisms that regulate dietary cholesterol absorption remain more poorly understood. One central component of this pathway is the Niemann-Pick C1-like 1 gene NPC1L1, which encodes a plasma membrane protein that mediates the uptake of dietary cholesterol by the intestine (Wang 2007;Ge et al. 2008). Mouse mutants for NPC1L1 display significantly reduced levels of cholesterol absorption and are insensitive to treatment with the anti-hypercholesterolemia drug ezetimibe, which acts as a specific NPC1L1 inhibitor (Davis et al. 2008). Another major regulator of cholesterol homeostasis is the liver X receptor a (LXRa) nuclear receptor, which binds cholesterol metabolites and regulates the transcription of genes that control cholesterol transport and metabolism, including NPC1L1 (Duval et al. 2006;Kalaany and Mangelsdorf 2006;Valasek et al. 2007).We used the fruit fly, Drosophila, as a model system to study the regulation of cholesterol homeostasis. Unlike vertebrates, insects are cholesterol auxotrophs that are unable to synthesize this essential compound (Va'nt Hoog 1936). Little is known, however, about the mechanisms that regulate the uptake of dietary cholesterol in Drosophila. A recent study showed that the fly ortholog of NPC1L1, npc1b, is expressed specifically in the midgut and is essential for dietary cholesterol absorption (Voght et al. 2007). Other NPC disease gene homologs in Drosophila also contribute to cholesterol homeostasis. The Drosophila ortholog of vert...

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Section: Dhr96 Binds Cholesterolmentioning

confidence: 48%

The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis

Horner¹,

Pardee²,

et al. 2009

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“…In addition to its role in cell membrane structure and function, cholesterol may regulate gene expression through its binding to a ligand-dependant nuclear receptor; recent studies have shown that cholesterol is a ligand of ROR␣, which permits its transcriptional activity. 1,2 This is the first assertion that native cholesterol can activate a transcription factor. Cholesterol derivatives (including steroid hormones and vitamin D) and oxidized cholesterol derivatives (oxysterols) are known to be signaling molecules, 29 -31 but this is the first time that native cholesterol has been proposed to activate a transcription factor.…”

Section: Cellular Roles Of Cholesterolmentioning

confidence: 99%

“…Kallen et al 1 were shown to be the major ligands present in the LBD. 2 ROR␣ LBD, expressed in Spodoptera frugiperda 9 (Sf-9) insect cells, was in a liganded form with bound cholesterol, which stabilizes the receptor in an agonistic conformation. 1 Depletion of cholesterol in U20S osteosarcoma cells using the statin lovastatin in LDL-free serum dramatically decreases the transcriptional activity of ROR␣, suggesting that changes in intracellular cholesterol level are capable of modulating the transcriptional activity of ROR␣, which implies that cholesterol is a "real" ligand rather than just a structural cofactor.…”

Section: Cellular Roles Of Cholesterolmentioning

confidence: 99%

“…1,2 ROR␣ has been shown to be implicated in the development and/or differentiation of many tissues, and provides protection against age-related degenerative processes, including atherosclerosis. In addition, ROR␣ activates the apolipoprotein A-I (apoA-I) and apoC-III gene transcription, 3,4 modulates the vasomotor tone of small resistance arteries, and participates in the regulation of postischemic angiogenesis.…”

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confidence: 99%

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The “CholesteROR” Protective Pathway in the Vascular System

Boukhtouche

Mariani

Tedgui

2004

ATVB

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Abstract-Retinoic acid receptor-related Orphan Receptor ␣ (ROR␣) is a member of the nuclear hormone receptor superfamily. ROR␣ has long been considered as a constitutive activator of transcription in the absence of exogenous ligand; however, cholesterol has recently been identified as a natural ligand of ROR␣. The spontaneous staggerer (sg/sg) mutation is a deletion in the Rora gene that prevents the translation of the ligand-binding domain (LBD), leading to the loss of ROR␣ activity. The homozygous Rora sg/sg mutant mouse, of which the most obvious phenotype is ataxia associated with cerebellar degeneration, also displays a variety of other phenotypes, including several vascular ones; in particular, dysfunction of smooth muscle cells and enhanced susceptibility to atherosclerosis. Moreover, ROR␣ appears to participate in the regulation of plasma cholesterol levels, and has been shown to positively regulate apolipoprotein (apo)A-I and apoC-III gene expression. Yet its activity is regulated by cholesterol itself, making ROR␣ an intracellular cholesterol target. Key Words: ROR␣ Ⅲ cholesterol Ⅲ statins Ⅲ atherosclerosis Ⅲ lipid homeostasis R etinoic acid receptor-related Orphan Receptor ␣ (ROR␣), initially described as an orphan nuclear receptor, has recently been deorphanized by the identification of its natural ligand as cholesterol or a cholesterol derivative. 1,2 ROR␣ has been shown to be implicated in the development and/or differentiation of many tissues, and provides protection against age-related degenerative processes, including atherosclerosis. In addition, ROR␣ activates the apolipoprotein A-I (apoA-I) and apoC-III gene transcription, 3,4 modulates the vasomotor tone of small resistance arteries, and participates in the regulation of postischemic angiogenesis. 5,6 This review focuses on the link between ROR␣, vascular biology, and cholesterol homeostasis. Interestingly, recent findings suggest that ROR␣ is an intracellular cholesterol target. 1

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“…Native state (nondenaturing) mass spectrometry can be used to determine the presence or absence of drug ligands and in some instances, used for affinity screening [10,11]. In the post-genomic era, nondenaturing mass spectrometry has been utilized to identify native ligands, endogenous ligands that activate or deactivate novel drug targets, for example orphan nuclear hormone receptors [12][13][14][15][16][17][18]. Typically, native state MS is used to detect the presence of the native ligand while additional MS experiments (GC/MS, exact mass LC/MS, etc.)…”

Section: Ass Spectrometry (Ms) Is a Powerful Techniquementioning

confidence: 99%

Automated in-line gel filtration for native state mass spectrometry

Waitt

Wisely

et al. 2008

J. Am. Soc. Mass Spectrom.

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Characterization of protein-ligand complexes by nondenaturing mass spectrometry provides direct evidence of drug-like molecules binding with potential therapeutic targets. Typically, protein-ligand complexes to be analyzed contain buffer salts, detergents, and other additives to enhance protein solubility, all of which make the sample unable to be analyzed directly by electrospray ionization mass spectrometry. This work describes an in-line gel-filtration method that has been automated and optimized. Automation was achieved using commercial HPLC equipment. Gel column parameters that were optimized include: column dimensions, flow rate, packing material type, particle size, and molecular weight cut-off. Under optimal conditions, desalted protein ions are detected 4 min after injection and the analysis is completed in 20 min. The gel column retains good performance even after Ͼ200 injections. A demonstration for using the in-line gel-filtration system is shown for monitoring the exchange of fatty acids from the pocket of a nuclear hormone receptor, peroxisome proliferator activator-␦ (PPAR␦) with a tool compound. Additional utilities of in-line gel-filtration mass spectrometry system will also be discussed. (J Am Soc Mass Spectrom 2008, 19, 239 -245)

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

Cited by 68 publications

References 38 publications

The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis

The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis

The “CholesteROR” Protective Pathway in the Vascular System

Automated in-line gel filtration for native state mass spectrometry

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