Unveiling hidden features of orphan nuclear receptors: The case of the small heterodimer partner (SHP)

Abstract

The small heterodimer partner (SHP) is an atypical nuclear receptor lacking the N-terminal ligand-independent activation domain and the DNA binding domain. SHP acts as transcriptional inhibitor of a large set of nuclear receptors, among which ER, AR, CAR, RXR, GR, LXR and ERRγ. The repression mechanism of SHP involves several actions including competition with coactivators binding on the AF-2 of nuclear receptors and recruitment of transcriptional inhibitors such as EID-1. The investigation of the structure and repression mechanism of SHP is a challenging task for a full understanding of nuclear receptor interaction pathways and functions. So far, mutational analyses in multiple populations identified loss of function mutants of SHP gene involved in mild obesity, increased birth weight and insulin levels. Furthermore, experimental mutagenesis has been exploited to characterize the interactions between SHP and the transcriptional inhibitor EID-1. With the aim of gaining insight into the structural basis of SHP repression mechanism, we modelled SHP and EID-1 structures. Docking experiments were carried out to identify the EID-1 binding surface on SHP structure. The results obtained in this study allow for the first time a unique interpretation of many experimental data available from the published literature. In addition, a fascinating hypothesis raises from the inspection of the proposed SHP structure: the presence of a potential unexpected ligand binding site, supported by recently available experimental data that may represent a breakthrough in the design and development of synthetic modulators of SHP functions.

Introduction

Nuclear receptor superfamily consist of 48 members including (i) the endocrine receptors whose endogenous ligands include thyroid and steroid hormones, derivatives and metabolites of Vitamins A and D; (ii) orphan nuclear receptors whose endogenous ligands are yet to be discovered [1].

In the last years, a number of orphan nuclear receptors have been adopted by the discovery of naturally occurring or synthetic ligands [2]. For these receptors, it has been possible to suggest a link to their physiological functions. In general terms, the physiological functions of nuclear receptors are dependent on the selective regulation of the expression of target genes that are involved in specific cellular processes such as cell growth, differentiation, development and homeostasis [3], [4].

All the members of the superfamily of nuclear receptors share a modular structure [4], [5], [6], [7]. In particular, they have a highly conserved central DNA-binding domain (DBD); a N-terminal domain which contains the ligand-independent transcriptional activation function-1 (AF-1); a C-terminal domain or ligand binding domain (LBD) endowed with the ligand-dependent transcriptional activation function-2 (AF-2). The DBD contains two cysteine coordinated zinc-fingers which are responsible for DNA binding and dimerization. In particular, DNA binding occurs in specific nucleotide sequences within the target gene promoters which are termed hormone response elements. The N-terminal domain varies in length and amino acid composition and it is responsible for the ligand-independent activation of receptor's transcription function through the interaction with other nuclear factors. The LBD contains the endogenous ligand binding site and it is involved in the homo/hetero-dimerization and in the recruitment of coregulators [8], [9]. In particular, the latter occurs through conformational shifts of the AF-2 helix (H12) of LBD upon ligand binding [10], [11].

The small heterodimer partner (SHP) is an atypical member of the above superfamily [12], [13]. Indeed, it lacks the ligand-independent activation domain and the DBD. Furthermore, no ligands, endogenous nor exogenous, have so far been identified. The expression of SHP gene is regulated by a number of nuclear receptors including bile acid receptor (FXR), steroidogenic factor-1 (SF-1), hepatocyte nuclear factor-4α (HNF-4α), liver receptor homolog-1 (LRH-1), estrogen receptor-α (ERα) and estrogen related receptor-γ (ERRγ) [14], [15], [16], [17].

In particular, the up-regulation of SHP gene by FXR plays a pivotal role in the control of bile acid metabolism and cholesterol homeostasis through the following SHP-dependent repression of cholesterol 7 alpha-hydroxylase gene (CYP7A1) [16], [18], [19]. Furthermore, the discovery of mutations of SHP gene that are associated with mild hyperinsulinemia and obesity, pinpoints a role of SHP in the control of glucose metabolism [20], [21].

The physiological effects of SHP occur through a direct interaction with a number of nuclear receptors and the following repression of their transcriptional activity. Hence, the control on the cholesterol homeostasis is achieved by the interaction of SHP with LRH-1 that positively regulates the expression of CYP7A1 [22]. In a similar way, glucose metabolism is controlled by the interaction of SHP with HNF-4α, a positive regulator of the insulin biosynthesis [23]. Other interaction partners of SHP are estrogen receptor (ER), androgen receptor (AR), constitutive androstane receptor (CAR), retinoid X receptor (RXR), liver X receptor (LXR), ERRγ, glucocorticoid receptor (GR), peroxisome proliferator-activated receptor-γ (PPARγ) [15], [24], [25], [26], [27]. Although SHP usually represses the transactivation of its interaction partners, it has been reported that SHP augments PPARγ transactivation [28]. Interestingly, the SHP mediated enhancement of the activity of PPARγ is linked to a direct interaction of SHP with the DBD of PPARγ. The repression mechanism of SHP involves at least two actions including: (i) competition with coactivators binding on the AF-2 of nuclear receptors and (ii) recruitment of unknown corepressors through its transrepression domain [23], [29], [30].

In particular, competition with coactivators binding on the AF-2 surface of interaction partners constitutes the molecular basis of the interaction between SHP and nuclear receptors. Briefly, the binding competition occurs through the interaction of three LxxLL-like motifs of SHP, also termed nuclear receptor (NR) boxes. Noteworthy, SHP was found to interact with ER through both NR boxes 1 and 2. [29] Conversely, the interaction of SHP with AR and GR is only mediated by NR box 2 [25], [26]. These results suggest that the redundancy of NR boxes of SHP is an essential feature for a selective interaction with diverse nuclear receptors.

The second action of SHP involves the recruitment of corepressors leading to the formation of a hetero-trimer complex with nuclear receptors. Båvner et al. reported that the E1A-like inhibitor of differentiation-1(EID-1) is recruited by SHP as corepressor [30]. EID-1 is a nuclear protein which blocks p300 coactivation function and it is involved in the inhibition of muscle differentiation [31], [32]. Interestingly, EID-1 does not interact directly with classical nuclear receptors, supporting the hypothesis of a hetero-trimer complex formation in which SHP acts as bridge between EID-1 and nuclear receptors.

According to Båvner and coworkers, the region encompassing residues 54–120 of EID-1 constitutes the SHP interaction domain and its relative binding surface is located on helices 3 and 12 of SHP. In addition, a recent report pinpointed that the loop region between helices 6 and 7 (residues 128–139) of SHP is also involved in defining the binding surface of the full length of EID-1 surface [32].

In the present study, we exploited homology modelling techniques to construct a 3D model of SHP. Furthermore, a protein fold was assigned to the SHP binding domain of EID-1 (residues 54–120) using ab initio structure prediction methods. The obtained structures were used to gain insight into the mechanism of SHP transcriptional repression. In particular, the recruitment of EID1 by SHP was studied in silico by mean of molecular docking experiments. The obtained results are discussed in the light of available experimental data. Being aware of the current limitations of the computational methods herein used, we thought that the investigation of such a model would help to clarify the repression mechanism of SHP and aid the design of potential modulators of SHP's functions.