Mechanisms that regulate chromosomal stability through the ubiquitin lipase activity of BRCA1

通过 BRCA1 泛素脂肪酶活性调节染色体稳定性的机制

• 批准号: 16591280
• 负责人: OHTA Tomohiko
• 金额: $ 2.18万
• 依托单位: St.Marianna University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16591280/
• 关键词: BRCA1; BARD1; familial breast cancer; ubiquitin ligase; RPB8; RING finger; nucleophosmin; Nucleophosmin

I have discovered in 2001 that BRCA1 constituted a RING heterodimer type ubiquitin ligase with BARD1. In this study I further investigated the substrates of BRCA1 to elacidate how the activity contributed the BRCA1's ability to maintain the chromosomal stability. I identified several substrates by our original screen methods. Among them I have investigated nucleophosmin/B23 (NPM) in 2004. BRCA1-BARD1 interacted with and polyubiquitinated NPM in vivo and in vitro. The polyubiquitination was non-traditional Lys-6-linked type, and therefore NPM was not degraded by the modification and instead stabilized. BRCA1-BARD1 and NPM were co-localized over the chromosomal surface during mitosis when NPM was actually ubiquitinated in vivo. I propose the lack of NPM ubiquitination could be the basis of centrosome hyper amplification and aneuploidy caused by BRCA1 deficiency. I next analyzed another candidate substrate, RPB8, in 2005. BRCA1-BARD1 interacted with and polyubiquitinated RPB8 in vivo and in vitro. The ubiquitination of RPB8 was observed 10 minutes after UV irradiation in HeLa cells stably expressing FLAG-tagged RPB8, and this ubiquitination was abolished by BRCA1 knockdown by siRNA. Interestingly HeLa cells stably expressing ubiquitin-resistant RPB8 mutant exhibited UV hypersensitivity. The results suggested that BRCA1 regulates chromosomal stability by transcription-coupled DNA repair through ubiquitination of RPB8.

我在2001年发现，BRCA1与BARD1共同构成了一种环状异二聚体型泛素连接酶。在这项研究中，我进一步研究了BRCA1的底物，以确定该活性如何有助于BRCA1‘S维持染色体稳定的能力。我通过我们最初的筛选方法鉴定了几种底物。其中，我在2004年对核磷脂/B23(NPM)进行了研究。在体内和体外，BRCA1-BARD1与NPM相互作用，并使NPM多泛素化。多泛素化是非传统的Lys-6连接类型，因此NPM不会被修饰降解，而是稳定下来。在有丝分裂过程中，当NPM在体内泛素化时，BRCA1-BARD1和NPM共定位于染色体表面。我认为缺乏NPM泛素化可能是BRCA1缺乏导致中心体过度扩增和非整倍体的基础。接下来，我在2005年分析了另一种候选基板RPB8。在体内和体外，BRCA1-BARD1与RPB8相互作用，并使RPB8多泛素化。紫外线照射10分钟后，在稳定表达FLAG标记的RPB8的HeLa细胞中观察到RPB8的泛素化，这种泛素化可被BRCA1被siRNA敲除。有趣的是，稳定表达泛素抗性RPB8突变体的HeLa细胞表现出紫外线超敏反应。这些结果表明，BRCA1通过RPB8泛素化的转录偶联DNA修复来调节染色体的稳定性。

项目成果

Breast tumor suppressor BRCA1

乳腺肿瘤抑制因子 BRCA1

• 发表时间: 2004
• 期刊: New Horizon Med. (Japanese) 36(4)
• 作者: Ohta T;Hayami R;Wu W;Fukuda M.
• 通讯作者: Fukuda M.

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage

• DOI: 10.1038/ncb1172
• 发表时间: 2004-10-01
• 期刊: NATURE CELL BIOLOGY
• 影响因子: 21.3
• 作者: Hu, J;McCall, CM;Xiong, Y
• 通讯作者: Xiong, Y

Down-regulation of BRCA1-BARD1 ubiquitin ligase by CDK2.

CDK2 下调 BRCA1-BARD1 泛素连接酶。

• 发表时间: 2005
• 期刊: Cancer Res. 65
• 作者: Hayami R;Sato K;Wu W;Nishikawa T;Hiroi J;Ohtani-Kaneko R;Fukuda M;Ohta T.
• 通讯作者: Ohta T.

Nucleophosmin/B23 is a candidate substrate for the BRCA1-BARD1 ubiquitin ligase

• DOI: 10.1074/jbc.c400169200
• 发表时间: 2004-07-23
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Sato, K;Hayami, R;Ohta, T
• 通讯作者: Ohta, T

A truncated splice bariant of human BARD1 that lacks the RING finger and ankyrin repeat.

人类 BARD1 的截短剪接体，缺少环指和锚蛋白重复序列​​。

• 发表时间: 2005
• 期刊: Cancer Lett. in press
• 作者: Nishida_M;Nagao T;Kurose H.(15);Bravou V. et al.;Xouri G. et al.;Sugimoto N. et al.;Nishitani H. et al.;西谷 秀男;Sei-ichi Tanuma;Sei-ichi Tanuma;Sei-ichi Tanuma;Sei-ichi Tanuma;Sei-ichi Tanuma;田沼 靖一;Sei-ichi Tanuma;Sei-ichi Tanuma;Sei-ichi Tanuma;Sei-ichi Tanuma;Sei-ichi Tanuma;Atsushi Yamauchi;Vinciane Regnier;Yoshikazu Mikami;深川竜郎;深川竜郎;Matsushita N;Makiko Tsuzuki
• 通讯作者: Makiko Tsuzuki