| 研究課題/領域番号 |
23KF0035
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| 研究機関 | 金沢大学 |
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研究代表者 |
篁 俊成 金沢大学, 医学系, 教授 (00324111)
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| 研究分担者 |
HEIN KO OO 金沢大学, 医学系, 外国人特別研究員
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| 研究期間 (年度) |
2023-04-25 – 2025-03-31
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| キーワード | Cysteine redoxome / Selenoprotein P / Reductive stress |
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| 研究実績の概要 |
For the selenoprotein P (SeP) mediated reductive stress target, we have identified more than 1500 significant cysteine residues that are oxidized or reduced under acute cold exposure in the brown adipose tissue (BAT) of WT and Selenop-knockout mice. Among them, 30 cysteine residues are reversible reactive cysteines. We are now focusing on the reactive cysteine of mic19, a member of MICOS, and evaluating its importance using in-silico and functional analysis.
For mRNA-binding proteins, we identified 17 proteins that specifically bind to Selenop mRNA by using the RiboTrap assay. One potential target is adck1 which is also involved in the assembly of MICOS. We have created Selenop mRNA and untranslated UUG-Selenop mRNA expression vector for further confirmation and functional analysis.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Currently, we only finished the comprehensive search for the target molecules in BAT samples because of the congestion in the LC-MS analysis workflow of our collaborator.
To express the various mutant variants and mRNA expression vectors in our in vitro assays, we are also trying to establish the immortalized mouse brown adipocyte cell line from the stromal vascular fraction cells of the BAT by using the SV40 vector. For in vivo analysis, we have also started the full-Selenop-mRNA-knockout mice, which are being crossbred with WT mice to obtain a higher genetic purity.
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| 今後の研究の推進方策 |
We will confirm the functional involvement of reactive cysteine of mic19 by expressing cysteine to alanine mutated variant in immortalized mouse brown adipocytes and check the mitochondria cristae formation using electron microscopy. Next, we will evaluate the regulatory mechanism of the target cysteine redox status and their involvement for the mitochondrial dynamics and functions by using in vitro and in vivo models.
We will also confirm the role of Selenop mRNA in mitochondrial functions by overexpressing the untranslated mRNA to the brown adipocytes. We will further confirm the binding site of mRNA and target proteins by using various mutant models of mRNA.
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| 次年度使用額が生じた理由 |
We have used the budget effectively for the LCMS and the in-silico analysis using gene ontology and molecular dynamic simulations. We will carry over the incurring amount and use it in combination with next year's budget for vector production for the various mutants of proteins and mRNAs, animal maintenance, electron microscopy & cryo-EM for the structural analysis of target proteins, and publication of the results.
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