investigating-protein-protein-interactions-live-cells-using

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Max Planck Institute for Psycholinguistics

Nuclear receptors (NRs) form a large superfamily of transcription factors that participate in virtually every key biological process. They control development, fertility, gametogenesis and are misregulated in many cancers. Their enormous functional plasticity as transcription factors relates in part to NR-mediated interactions with hundreds of coregulatory proteins upon ligand (e.g., hormone) binding to their ligand binding domains (LBD), or following covalent modification. Some coregulator association relates to the distinct residues that shape a coactivator binding pocket termed AF-2, a surface groove that primarily determines the preference and specificity of protein-protein interactions. However, the highly conserved AF-2 pocket in the NR superfamily appears to be insufficient to account for NR subtype specificity leading to fine transcriptional modulation in certain settings. Additional protein-protein interaction surfaces, most notably on their LBD, may contribute to modulating NR function. NR coregulators and chaperones, normally much larger than the NR itself, may also bind to such interfaces. In the case of the androgen receptor (AR) LBD surface, structural and functional data highlighted the presence of another site named BF-3, which lies at a distinct but topographically adjacent surface to AF-2. AR BF-3 is a hot spot for mutations involved in prostate cancer and androgen insensitivity syndromes, and some FDA-approved drugs bind at this site. Structural studies suggested an allosteric relationship between AF-2 and BF-3, as occupancy of the latter affected coactivator recruitment to AF-2. Physiological relevant partners of AR BF-3 have not been described as yet. The newly discovered site is highly conserved among the steroid receptors subclass, but is also present in other NRs. Several missense mutations in the BF-3 regions of these human NRs are implicated in pathology and affect their function in vitro. The fact that AR BF-3 pocket is a druggable site evidences its pharmacological potential. Compounds that may affect allosterically NR function by binding to BF-3 open promising avenues to develop type-specific NR modulators.

A conserved surface on the ligand binding domain of nuclear receptors for allosteric control.

Nuclear orphan receptor TLX (NR2E1) functions primarily as a transcriptional repressor and its pivotal role in brain development, glioblastoma, mental retardation and retinopathologies make it an attractive drug target. TLX is expressed in the neural stem cells (NSCs) of the subventricular zone and the hippocampus subgranular zone, regions with persistent neurogenesis in the adult brain, and functions as an essential regulator of NSCs maintenance and self-renewal. Little is known about the TLX social network of interactors and only few TLX coregulators are described. To identify and characterize novel TLX-binders and possible coregulators, we performed yeast-two-hybrid (Y2H) screens of a human adult brain cDNA library using different TLX constructs as baits. Our screens identified multiple clones of Atrophin-1 (ATN1), a previously described TLX interactor. In addition, we identified an interaction with the oncoprotein and zinc finger transcription factor BCL11A (CTIP1/Evi9), a key player in the hematopoietic system and in major blood-related malignancies. This interaction was validated by expression and coimmunoprecipitation in human cells. BCL11A potentiated the transrepressive function of TLX in an in vitro reporter gene assay. Our work suggests that BCL11A is a novel TLX coregulator that might be involved in TLX-dependent gene regulation in the brain.

Sara B. Estruch

Language and Genetics Department

Max Planck Institute for Psycholinguistics

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Sara B. Estruch

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Language and Genetics Department

Max Planck Institute for Psycholinguistics

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Language and Genetics Department