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Presence of functional domains in NADPH-dependent cytosolic 3,5,3'-Triiodo-L-thyronine (T3)-binding protein (p38CTBP)
S Suzuki1, J-I Mori, M Kobayashi
1Department of Aging Medicine and Geriatrics, Institute on Aging and Adaptation, Shinshu University Graduate School, Matsumoto, Nagano, Japan. soutaro@hsp.md.shinshu-u.ac.jp
Abstract:
Functional domains required for NADPH-binding, T(3)-binding, protein dimerization and cytosolic retention were analyzed in NADPH-dependent cytosolic 3,5,3'-triiodothyronine (T(3))-binding protein (p38CTBP) by using the deletion mutants. Wild-type p38CTBP (amino acids; 1-314) and a series of deletion mutants (amino acids; 1-79, 1-128, 1-146, 1-216, 37-314, and 1-145 with 270-314) were bacterially induced. NADPH-dependent T(3)-binding activity was not observed in all mutant p38CTBPs studied, although wild-type p38CTBP showed high-affinity T(3)-binding activity. Wild-type p38CTBP was able to bind a CL-6B column, none of the mutant p38CTBPs showed any binding activity. Pull-down analyses demonstrated that two regions between amino acid 128 and 146, and between 216 and 270, both of which possess helical structures, were required for homodimeric p38CTBP binding. In fluoroscopic studies, GFP-tagged p38CTBP was preferentially observed in cytoplasm. However, C-terminal region-deleted p38CTBP(1-216) was not only observed in cytoplasm, but also in nucleus. These results suggest that 1) multiple regions in p38CTBP molecule are required for T(3)-binding and NADPH binding, 2) two helical structures in p38CTBP molecule may be important in the homodimer formation, and 3) C-terminal region of p38CTBP contains the function to preserve the protein in cytoplasm.
Insights
This study identifies key regions of the NADPH-dependent cytosolic 3,5,3'-triiodothyronine (T(3))-binding protein (p38CTBP) essential for T(3) binding, dimerization, and cellular localization. Findings reveal multiple domains are crucial for these functions.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The cytosolic 3,5,3 '-triiodothyronine (T(3))-binding protein (p38CTBP) plays a role in cellular processes.
- Understanding the functional domains of p38CTBP is crucial for elucidating its mechanism of action.
Purpose of the Study:
- To delineate the functional domains of p38CTBP responsible for NADPH-binding, T(3)-binding, protein dimerization, and cytosolic retention.
- To analyze the structure-function relationship of p38CTBP using deletion mutants.
Main Methods:
- Bacterial expression and induction of wild-type and deletion mutant p38CTBP.
- NADPH-dependent T(3)-binding assays and CL-6B column chromatography.
- Pull-down assays to identify dimerization interfaces.
- Fluorescence microscopy using GFP-tagged proteins to determine subcellular localization.
Main Results:
- Wild-type p38CTBP exhibited high-affinity T(3)-binding and NADPH-binding activity, which was lost in all deletion mutants.
- Two helical regions (amino acids 128-146 and 216-270) were identified as essential for p38CTBP homodimerization.
- Deletion of the C-terminal region resulted in p38CTBP translocation from the cytoplasm to the nucleus, indicating its role in cytosolic retention.
Conclusions:
- Multiple distinct regions within p38CTBP are required for its T(3) and NADPH binding capabilities.
- Specific helical structures within p38CTBP are critical for mediating homodimer formation.
- The C-terminal domain of p38CTBP is essential for maintaining its localization within the cytoplasm.
