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Time and spacial analyses for mechanisms of intracellular traffic and localization of nuclear receptors using imaging analyses including FRET

使用成像分析（包括 FRET）对细胞内运输机制和核受体定位进行时间和空间分析

基本信息

批准号：
16590909
负责人：
KAWATE Hisaya
金额：
$ 2.3万
依托单位：
KYUSHU UNIVERSITY
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

项目摘要

I. Analyses of intracellular dynamics of mutant androgen receptors (AR) derived from two androgen insensitivity syndrome (AIS) patients.Intracellular dynamics of two different AR mutants (AR-C579F, AR-F582Y) derived from AIS patients were analysed using GFP-fused mutant ARs. The wild-type AR was ligand-dependently translocated into the nucleus where it formed fine subnuclear foci. Surprisingly, after the addition of dihydrotestosterone (DHT), the two mutant ARs initially formed large cytoplasmic dots, many of which were found to be close to mitochondria by electron microscopy. Subsequently, a proportion of the ligand-bound mutant ARs gradually entered the nucleus to form a smaller number of larger dots compared with the wild-type AR. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the intranuclear mobilities of the mutant ARs were decreased compared with that of the wild-type AR. These results suggest that the abnormal translocation, localization and mobility o … More f the mutant ARs may be the cause of AIS in these subjects.II. Analyses of transcriptional repression mechanisms by novel corepressors, TZF and TobWe showed that testicular zinc finger protein, TZF, was a novel corepressor for AR. TZF was ligand-dependently colocalized with AR and interacted with the N-terminus of AR. A central portion (amino acids 512-663) of TZF, TZF(512-663), is responsible for both binding to AR and repressing the transactivation. TZF recruited endogenous histone deacetylase 2 (HDAC2) and formed a complex with agonist-bound AR. Imaging analyses showed that TZF and TZF(512-663) were recruited by AR and simultaneously impaired distinct AR foci formation. Quantification of the foci number using a three-dimensional imaging method revealed that the number of intranuclear AR foci was related to its transactivation activity. Moreover, increased levels of TZF dissociated a coactivator, TIF2, from the AR foci and vice versa. These results indicate that the ligand-dependent transactivation function of AR is quantitatively related to its intranuclear foci formation, and suggest that corepressors, such as TZF, act on these intranuclear events competitively with coactivators.We also found that Tob, which was regarded as a negative regulator of osteoblast function, repressed transactivation function of AR, ER and GR. Less

I.两例雄激素不敏感综合征（AIS）患者雄激素受体（AR）突变体的细胞内动力学分析采用GFP融合的AR突变体，分析了AIS患者AR突变体（AR-C579 F，AR-F582 Y）的细胞内动力学。野生型AR以配体依赖性方式移位到细胞核中，在那里形成精细的亚核灶。令人惊讶的是，在加入双氢睾酮（DHT）后，这两种突变AR最初形成了大的细胞质点，通过电子显微镜发现其中许多点靠近线粒体。随后，一部分配体结合的突变AR逐渐进入细胞核，形成与野生型AR相比数量较少的较大点。荧光恢复后光漂白（FRAP）分析表明，突变体AR的核内迁移率降低与野生型AR相比。这些结果提示，在正常人外周血淋巴细胞中， ...更多信息突变型AR可能是这些受试者AIS的原因。对新型辅阻遏物TZF和TobWe转录抑制机制的分析表明，睾丸锌指蛋白TZF是AR的新型辅阻遏物。TZF与AR共定位，并与AR的N-末端相互作用。TZF的中心部分（氨基酸512-663）TZF（512-663）负责与AR结合并抑制反式激活。TZF募集内源性组蛋白去乙酰化酶2（HDAC 2），并与激动剂结合的AR形成复合物。成像分析显示TZF和TZF（512-663）被AR募集，同时损害不同AR灶的形成。使用三维成像方法定量的病灶数显示，核内AR病灶的数量与其反式激活活性有关。此外，TZF水平的增加使共激活因子TIF 2与AR灶分离，反之亦然。这些结果表明，AR的配体依赖性反式激活功能与其核内病灶的形成有定量关系，并提示辅抑制因子如TZF与辅激活因子竞争性地作用于这些核内事件，我们还发现Tob作为成骨细胞功能的负调节因子，抑制AR、ER和GR的反式激活功能。