Study Of The Interaction Between Nuclear Receptors And IMP?

Nuclear receptor superfamily is a kind of transcription factors regulated by the small molecule ligands,which can regulate the expression of downstream target genes,thus plays an important role in cell growth and differentiation,development and metabolism.These gene regulations have a close relationship with a series of diseases,such as diabetes,cancer,inflammation,and cardiovascular disease,etc.As a result,the nuclear receptors have already become key targets in drug discovery.Since the importance of nuclear receptors and their functional activities in the nucleus,it is a very significant cycle for the regulation of signaling pathway for their transport from cytoplasm to nucleus after synthesized in cytoplasm.Although much more researches have been performed in the functional and regulation mechanism of nuclear receptor recently,it still remains unknown for the mechanism of their nuclear import pathway.The entry into the nucleus of nucleoprotein is regulated mainly by the classical nuclear transport pathway mediated by IMP ?/?,while the nuclear input receptor IMP a serves as a connector with the NLS of nucleoprotein and IMP ?(or IMP beta homologues)via its different ends,respectively.In order to figure out that if the nuclear receptor enters into nucleus by binding of its NLS and IMP a,we constructed the expression plasmids which contains fragments of IMP as or nuclear receptors by cloning method,and detected the interaction between IMP a and nuclear receptors by His-tag pull-down analysis.As a result,we found that the nuclear receptor ER a,THR al,and FXR interacted with different subtypes of IMP a,which suggests that the subtypes of IMP a are interchangeable when they trasport the nuclear receptors into the nucleus.By Co-Immunoprecipitation assay in vivo and molecular sieve chromatography assay in vitro,we have validated the interaction of ER ? and THR ?1 with IMP a,respectively,and obtained their complex with high quality for further crystallization.At the same time,FXR and IMP a showed a weak interaction.We choose the ER a/IMP as and THR al/IMP as to crystallize because of their high expression level and strong binding capacity,and got the initial crystals of ER ? and IMP as.In a word,we discovered and verified the interaction of ER a and THR al with IMP a,and crystallized the complexes.Our findings have a great guiding significance for the molecular mechanism of interaction between nuclear receptors and IMP a,and provide a unique perspective of exploring the mechanism of nuclear receptors into the nucleus and also a scientific basis for non-ligand medicine research targeting nuclear receptors.