Michael Albers

Identification of farnesoid X receptor beta as a novel mammalian nuclear receptor sensing lanosterol

ABSTRACT Nuclear receptors are ligand-modulated transcription factors. On the basis of the completed human genome sequence, this family was thought to contain 48 functional members. However, by mining human and mouse genomic sequences, we identified FXRβ as a novel family member. It is a functional receptor in mice, rats, rabbits, and dogs but constitutes a pseudogene in humans and primates. Murine FXRβ is widely coexpressed with FXR in embryonic and adult tissues. It heterodimerizes with RXRα and stimulates transcription through specific DNA response elements upon addition of 9-cis-retinoic acid. Finally, we identified lanosterol as a candidate endogenous ligand that induces coactivator recruitment and transcriptional activation by mFXRβ. Lanosterol is an intermediate of cholesterol biosynthesis, which suggests a direct role in the control of cholesterol biosynthesis in nonprimates. The identification of FXRβ as a novel functional receptor in nonprimate animals sheds new light on the species differences in cholesterol metabolism and has strong implications for the interpretation of genetic and pharmacological studies of FXR-directed physiologies and drug discovery programs.

Automated Yeast Two-hybrid Screening for Nuclear Receptor-interacting Proteins

High throughput analysis of protein-protein interactions is an important sector of hypothesis-generating research. Using an improved and automated version of the yeast two-hybrid system, we completed a large interaction screening project with a focus on nuclear receptors and their cofactors. A total of 425 independent yeast two-hybrid cDNA library screens resulted in 6425 potential interacting protein fragments involved in 1613 different interaction pairs. We show that simple statistical parameters can be used to narrow down the data set to a high confidence set of 377 interaction pairs where validated interactions are enriched to 61% of all pairs. Within the high confidence set, there are 64 novel proteins potentially binding to nuclear receptors or their cofactors. We discuss several examples of high interest, and we expect that communication of this huge data set will help to complement our knowledge of the protein interaction repertoire of this family of transcription factors and instigate the characterization of the various novel candidate interactors.

A Novel Principle for Partial Agonism of Liver X Receptor Ligands COMPETITIVE RECRUITMENT OF ACTIVATORS AND REPRESSORS

Partial, selective activation of nuclear receptors is a central issue in molecular endocrinology but only partly understood. Using LXRs as an example, we show here that purely agonistic ligands can be clearly and quantitatively differentiated from partial agonists by the cofactor interactions they induce. Although a pure agonist induces a conformation that is incompatible with the binding of repressors, partial agonists such as GW3965 induce a state where the interaction not only with coactivators, but also corepressors is clearly enhanced over the unliganded state. The activities of the natural ligand 22(R)-hydroxycholesterol and of a novel quinazolinone ligand, LN6500 can be further differentiated from GW3965 and T0901317 by their weaker induction of coactivator binding. Using biochemical and cell-based assays, we show that the natural ligand of LXR is a comparably weak partial agonist. As predicted, we find that a change in the coactivator to corepressor ratio in the cell will affect NCoR recruiting compounds more dramatically than NCoR-dissociating compounds. Our data show how competitive binding of coactivators and corepressors can explain the tissue-specific behavior of partial agonists and open up new routes to a rational design of partial agonists for LXRs.

Ubiquitin-interaction motifs of RAP80 are critical in its regulation of estrogen receptor α

In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERα) in an agonist-dependent manner. The interaction is specific for ERα as ERβ and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERα was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERα is sufficient for interaction with RAP80. RAP80 overexpression reduces ERα polyubiquitination, increases the level of ERα protein, and enhances ERα-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERα protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERα, but eliminates the effects of RAP80 on ERα polyubiquitination, the level of ERα protein, and ERα-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERα as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERα activity.In this study, we demonstrate that receptor-associated protein 80 (RAP80) interacts with estrogen receptor alpha (ERα) in an agonist-dependent manner. The interaction is specific for ERα as ERβ and several other nuclear receptors tested did not interact with RAP80. Interaction between RAP80 and ERα was supported by mammalian two-hybrid, GST pull-down, and co-immunoprecipitation analyses. The hinge/ligand-binding domain of ERα is sufficient for interaction with RAP80. RAP80 overexpression reduces ERα polyubiquitination, increases the level of ERα protein, and enhances ERα-mediated transactivation. Knockdown of endogenous RAP80 expression by small-interfering RNA (siRNA) reduced ERα protein level and the E2-dependent induction of pS2. In this study, we also demonstrate that RAP80 contains two functional ubiquitin-interaction motifs (UIMs) that are able to bind ubiquitin and to direct monoubiquitination of RAP80. Deletion of these UIMs does not affect the ability of RAP80 to interact with ERα, but eliminates the effects of RAP80 on ERα polyubiquitination, the level of ERα protein, and ERα-mediated transcription. These data indicate that the UIMs in RAP80 are critical for the function of RAP80. Our study identifies ERα as a new RAP80-interacting protein and suggests that RAP80 may be an important modulator of ERα activity.

Pharmacologic modulation of RORγt translates to efficacy in preclinical and translational models of psoriasis and inflammatory arthritis

The IL-23/IL-17 pathway is implicated in autoimmune diseases, particularly psoriasis, where biologics targeting IL-23 and IL-17 have shown significant clinical efficacy. Retinoid-related orphan nuclear receptor gamma t (RORγt) is required for Th17 differentiation and IL-17 production in adaptive and innate immune cells. We identified JNJ-54271074, a potent and highly-selective RORγt inverse agonist, which dose-dependently inhibited RORγt-driven transcription, decreased co-activator binding and promoted interaction with co-repressor protein. This compound selectively blocked Th17 differentiation, significantly reduced IL-17A production from memory T cells, and decreased IL-17A- and IL-22-producing human and murine γδ and NKT cells. In a murine collagen-induced arthritis model, JNJ-54271074 dose-dependently suppressed joint inflammation. Furthermore, JNJ-54271074 suppressed IL-17A production in human PBMC from rheumatoid arthritis patients. RORγt-deficient mice showed decreased IL-23-induced psoriasis-like skin inflammation and cytokine gene expression, consistent with dose-dependent inhibition in wild-type mice through oral dosing of JNJ-54271074. In a translational model of human psoriatic epidermal cells and skin-homing T cells, JNJ-54271074 selectively inhibited streptococcus extract-induced IL-17A and IL-17F. JNJ-54271074 is thus a potent, selective RORγt modulator with therapeutic potential in IL-23/IL-17 mediated autoimmune diseases.

Regulation of IL-22BP in psoriasis

IL-22 is a potent pro-inflammatory cytokine upregulated in psoriasis and in other inflammatory diseases. The function of IL-22 is regulated by the soluble scavenging receptor, IL-22 binding protein (IL-22BP or IL-22RA2). However, the role and regulation of IL-22BP itself in the pathogenesis of inflammatory disease remain unclear. We used the TLR7 agonist Imiquimod (IMQ) to induce a psoriasis-like skin disease in mice and found a strong downregulation of IL-22BP in the affected skin as well as in the lymph nodes of animals treated with IMQ. We also analysed psoriatic skin of patients and compared this to skin of healthy donors. Interestingly, IL-22BP expression was similarly downregulated in skin biopsies of psoriasis patients compared to the skin of healthy donors. Since IL-22BP is expressed foremost in dendritic cells, we characterized its expression in monocyte-derived dendritic cells (MoDC) during maturation. In this way, we found Prostaglandin E2 (PGE2) to be a potent suppressor of IL-22BP expression in vitro. We conclude that regulation of IL-22BP by inflammatory mediators is an important step for the progression of inflammation in the skin and possibly also in other autoimmune diseases.