Structure and function of proteins regulating cell growth and differentiation during asexual reproduction of tunicates

被囊类动物无性繁殖过程中细胞生长和分化调节蛋白的结构和功能

• 批准号: 11480221
• 负责人: KAWAMURA Kazuo
• 金额: $ 3.65万
• 依托单位: Kochi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480221/
• 关键词: tunicate; budding; multipotency; retinoic acid; retinoid X receptor; serine protease; serine protease inhibitor; lectin; αヘリックス; 無性生殖; セリンプロテアーゼ; プロテアーゼインヒビター; C型レクチン; 上皮; アルデヒドデヒドロゲナーゼ; イチノイン酸受容体; レチノイドX受容体; プロテアーゼ

Budding tunicates possess a remarkable potential of tissue renewal. Interestingly, this renewal is not accomplished by undifferentiated stem cell but by differentiated somatic cells. As soon as budding or regeneration begins, pigmented epithelial cells (atrial epithelium) undergo dedifferentiation, enter cell division cycle and take undifferentiated shape. This kind of morphogenesis that accompanies dedifferentiation and/or transdifferentiation is also known in Cnidaria and Annelida. In contrast with vertebrate stem cell system, we call this as 'primitive' stem cell system. The goal of our project is to disclose structure and function of proteins that regulate cell growth and differentiation of multipotent cells in budding tunicates.First we have looked for genes that are expressed specifically at budding phase by the method of subtraction, differential display and EST analysis. Secondly, spatio-temporal expression of those genes has been examined by means of in situ hybridization and … More immunohistochemistry. Thirdly, in order to elucidate gene functions, recombinant proteins and mutant proteins have been produced and assayed in vitro using tunicate cell lines.In this project, we have cloned eight kinds of budding-specific genes {retinoid X receptor (RXR), aldehyde dehydrogenase (ALDH), serine protease (TRAMP), serine protease inhibitor (SERPIN), lectin (TC14), reverse transcriptase (RT), cytochrome b5 (Cb5), mitochondrial 16S ribosomal RNA (MTlrRNA)}. ALDH is expressed in the epidermis very early during budding or regeneration. It plays a role in biosynthesis of retinoic acid. Then, retinoic acid induces production and excretion of TRAMP and SERPIN that are dedifferentiation factors of the multipotent cells in budding tunicate. TC14 counteracts the dedifferentiation factors, and its activity is canceled with ease by inhibition sugar such as galactose. Cb5 is known to plays a role in fatty acid biosynthesis. In budding tunicates, fatty acids show cell growth-promoting activity. RT is expressed in the multipotent epithelium. As it resembles fruit fly retroposon, gypsy, RT may play a role in rejuvenation of multipotent, but differentiated cells in budding tunicates. Less

出芽的被囊动物具有显著的组织更新潜力。有趣的是，这种更新不是由未分化的干细胞完成的，而是由分化的体细胞完成的。一旦出芽或再生开始，色素上皮细胞（心房上皮）就会进行去分化，进入细胞分裂周期，形成未分化的形态。这种伴随去分化和/或转分化的形态发生在刺胞动物和环节动物中也有。与脊椎动物干细胞系统相比，我们称之为“原始”干细胞系统。我们项目的目标是揭示出芽被囊中调节细胞生长和多能细胞分化的蛋白质的结构和功能。首先，我们通过减法、差异显示和EST分析的方法寻找在出芽期特异性表达的基因。其次，通过原位杂交和免疫组化检测了这些基因的时空表达。第三，为了阐明基因的功能，利用被囊细胞系制备了重组蛋白和突变蛋白，并进行了体外实验。在本项目中，我们克隆了8种发芽特异性基因{类视黄酮X受体（RXR）、醛脱氢酶（ALDH）、丝氨酸蛋白酶（TRAMP）、丝氨酸蛋白酶抑制剂（SERPIN）、凝集素（TC14）、逆转录酶（RT）、细胞色素b5 （Cb5）、线粒体16S核糖体RNA (MTlrRNA)}。ALDH在萌芽或再生的早期表达于表皮。它在维甲酸的生物合成中起作用。然后，维甲酸诱导出芽被囊多能细胞的去分化因子TRAMP和SERPIN的产生和排泄。TC14可以抵消去分化因子，其活性容易被半乳糖等抑制糖抵消。已知Cb5在脂肪酸生物合成中起作用。在出芽的被囊动物中，脂肪酸表现出促进细胞生长的活性。RT在多能上皮中表达。由于它类似于果蝇反转录子，gypsy， RT可能在出芽被囊中多能分化细胞的返年轻化中起作用。少

项目成果

Matsumoto, J., Nakamoto, C., Fujiwara, S., Yubisui, T. and Kawamura, K.: "A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation in budding tunicates"Development. 128. 3339-3347 (2001)

Matsumoto, J.、Nakamoto, C.、Fujiwara, S.、Yubisui, T. 和 Kawamura, K.：“一种新型 C 型凝集素调节出芽被囊动物的细胞生长、细胞粘附和细胞分化”开发。

Kamimura,M.: "Functional retinoid receptors in budding ascidians."Develop.Growth Differ.. 42. 18 (2000)

Kamimura,M.：“出芽海鞘中的功能性视黄醇受体。”Develop.Growth Differ.. 42. 18 (2000)

Matsumoto, J., et al.: "A novel C-type lectin regulating cell growth, cell adhesion and cell differentiation in budding tunicates"Development. 128. 3339-3347 (2001)

Matsumoto, J. 等人：“一种新型 C 型凝集素调节出芽被囊动物的细胞生长、细胞粘附和细胞分化”。

Ohashi, M., et al.: "A retinoic acid-inducible modular protease in budding ascidians"Developmental Biology. 214. 38-45 (1999)

Ohashi, M. 等人：“海鞘出芽中的视黄酸诱导型模块化蛋白酶”发育生物学。

Yubisui, T., Takahashi, H., Takabayashi, S., Fujiwara, S., Kawamura, K.: "Characterization of cytochrome b5 in the ascidian, Polyandrocarpa misakiensis and budding-specific expression"Journal of Biochemistry. 129. 709-716 (2001)

Yubisui, T.、Takahashi, H.、Takabayashi, S.、Fujiwara, S.、Kawamura, K.：“海鞘、Polyandrocarpa missakiensis 中细胞色素 b5 的表征和出芽特异性表达”生物化学杂志。