A structural signature motif enlightens the origin and diversification of nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the α-helix, called π-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the π-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the π-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the π-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the π-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the π-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors.

Profiling of gene-dependent translational progress in cell-free protein synthesis by real-space imaging.

In general, gene-dependent translational progress affects the efficiency of protein expression. To evaluate the translational progress of protein synthesis, it is necessary to trace the time course of translation as well as the quantity of products. Here we present a new method for tracking translation steps in cell-free protein synthesis using atomic force microscopy (AFM). The cell-free protein synthesis system is useful to track the inherent translational progress of a target gene, whereas conventional UV absorption measurement coupled with density gradient fractionation is difficult to analyze such small sample quantities. Because the high resolution of AFM enables us to clearly count the number of ribosomes included in polysomes, polysome profiles can be obtained directly without complicated fractionation. With this method, we could elucidate the detailed polysome profile with only 1 microl of sample solution. We observed the translational progress of green fluorescent protein synthesis, a model of high-expression protein, as well as human retinoid X receptor. Detailed polysome profiles showed different patterns of translational progress and were clearly associated with the results of time-dependent protein expression. Our study suggests the possibility for comprehensive character analysis of inherent gene-dependent translational progress.

Expression of retinoid receptors in multiple cell lineages in the gastric mucosae of mice and humans.

BACKGROUND AND AIM: In mice and humans, the gastric epithelial progenitors undergo proliferation and bipolar migration from the isthmus associated with their differentiation into mucus-, acid- and pepsinogen-secreting cell lineages. Little is known about factors that control the dynamics of these isthmal progenitor cells. Retinoids have long been known as chemopreventive agents against gastric mucosal damage and carcinogenesis. The aim of the present study was to examine the cellular localization of the various retinoid receptors proteins (RAR and RXR) in the gastric epithelium of mice and humans. METHODS: Gastric antral biopsies of normal individuals and the oxyntic and antral regions of the mouse stomach were processed for immunohistochemistry using anti-RAR and anti-RXR antibodies. To label the progenitor cell zone, some sections were also probed with antibodies specific for proliferating cell nuclear antigen. RESULTS: The immunoprobed oxyntic mucosal sections of the mice showed that RXRbeta protein was present in the epithelial isthmal cells, neck cells, zymogenic cells and some pit and parietal cells. In addition, RARbeta was found in isthmal and neck cells, and RARgamma was mainly found in neck cells. In the mouse antrum, only RXRbeta was detected in the isthmal cells and their pit and gland cell descendents. In humans, immunoprobed antral sections showed that RARbeta, RARgamma, RXRalpha and RXRgamma proteins are expressed in the isthmal, pit and gland cells. CONCLUSIONS: Retinoid receptors are expressed in multiple cell lineages of the mouse and human gastric epithelium and may, therefore, account for the possible effects of retinoids on gastric epithelial cell proliferation and differentiation.