Secretory expression of thyroid hormone receptor using transgenic silkworms and its DNA binding activity.

Silkworms are economically important insects that have the ability to produce large amounts of silk. They have mass breeding methods and silk glands, which are specialized tissues that secrete silk fibroin and sericin. Thus, the production of recombinant proteins in a transgenic silkworm system is a promising approach. We developed a silkworm, Bombyx mori, as a host expression insect for recombinant proteins and successfully produced different proteins including antibodies, glycoproteins, and membrane receptors. The thyroid hormone receptor (TR) is a regulatory factor for many physiological phenomena. It is a lipophilic protein that has DNA-binding and ligand-binding domains. Based on our previous experiences, it was inferred that the recombinant TR easily formed aggregates and precipitates which is potentially due to an unstructured hinge domain. We applied the silkworm expression system to produce mice TRß1 that was fused with glutathione S-transferase. Using 160 larvae, the yield of the recombinant GST-TRß was approximately 4 mg, and the purified GST-TRß completely retained its physiological activity. Our results indicated that the recombinant TRß was secreted extracellularly using the silk fibroin signal peptide sequence. Moreover, we found that the expression system of silkworms was applicable to nuclear proteins.

Molecular characterization of human thyroid hormone receptor ß isoform 4.

Thyroid hormone exerts a pleiotropic effect on development, differentiation, and metabolism through thyroid hormone receptor (TR). A novel thyroid hormone receptor ß isoform (TRß4) was cloned using PCR from a human pituitary cDNA library as a template. We report here the characterization of TRß4 from a molecular basis. Temporal expression of TRß4 during the fetal period is abundant in the brain and kidney, comparable with the adult pattern. Western blot analysis revealed that TRs are ubiquitination labile proteins, while TRß1 is potentially stable. TRß1, peroxisome proliferator-activated receptors (PPAR), and vitamin D receptor (VDR), which belong to class II transcription factors that function via the formation of heterodimeric complexes with retinoid X receptor (RXR), were suppressed by TRß4 in a dose-dependent manner. Thus, TRß4 exhibits ligand-independent transcriptional silencing, possibly as a substitute for dimerized RXR. In this study, TRß1 and TRß4 transcripts were detected in several cell lines. Quantitative RT-PCR assay showed that the expression of TRß4 in human embryonic carcinoma cells of the testis was suppressed by sex hormone in a reciprocal manner to TRß1. In contrast, TRß4 was expressed under a high dose of triiodothyronine (T3) in a reciprocal manner to TRß1. Finally, in transiently transfected NIH-3T3 cells, green fluorescence protein (GFP)-tagged TRß4 was mostly nuclear in both the absence and the presence of T3. By mutating defined regions of both TRßs, we found that both TRß1 and TRß4 had altered nuclear/cytoplasmic distribution as compared with wild-type, and different to T3 and the nuclear receptor corepressor (NCoR). Thus, site-specific DNA binding is not essential for maintaining TRßs within the nucleus.

Structural determinants of selective thyromimetics.

The thyromimetic GC-1 shows a preference for binding the beta form of the thyroid hormone receptor (TR). GC-1 was designed as an analogue of the thyromimetic DIMIT, which has a lower affinity for TR and is not selective. GC-1 has a methylene group linking its two aromatic rings and an oxyacetic acid polar side chain, while DIMIT has an ether oxygen linking its aromatic rings and an l-alanine polar side chain. The structural features of GC-1 that confer its greater affinity and selectivity compared to DIMIT were analyzed with the preparation of analogues that bear only one of their two different structural features. The analogue of GC-1 with a biaryl ether has selectivity comparable to that of GC-1, while the analogue of DIMIT with a methylene group linking its aromatic rings is only slightly selective. These results demonstrate that the oxyacetic acid side chain of GC-1 is critical in conferring TR-beta selectivity.