REGULATION AND FUNCTION OF P63

P63的调节和功能

• 批准号: 8170928
• 负责人: ZHI-XIONG Jim XIAO
• 金额: $ 0.11万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170928
• 关键词: Alternative Splicing; Apical Ectodermal Ridge; Birth; Breast; C-terminal; Chimeric Proteins; Cloning; Computer Retrieval of Information on Scientific Projects Database; DNA Binding Domain; Defect; Development; Epidermis; Epithelial; Failure; Family member; Funding; Genes; Grant; Hand; Hela Cells; Homologous Gene; Human; Individual; Inherited; Institution; Isopropyl Thiogalactoside; Limb structure; Mammary gland; Maps; Mass Spectrum Analysis; Mesenchymal; Mus; N-terminal; Plasmids; Play; Prostate; Protein Family; Protein Isoforms; Proteins; Regulation; Research; Research Personnel; Resources; Role; Salivary Glands; Source; Staining method; Stains; Structure; Syndrome; TP53 gene; Tail; Tissues; Transactivation; United States National Institutes of Health; appendage; craniofacial; foot; lacrimal; malformation; orofacial; promoter; protein complex; tumorigenesis; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. p63, a p53 homologue, is transcribed into six isoforms derived from differential promoter usage and alternative splicing. The transactivation (TA) isoforms, which resemble p53, are generated by the use of an upstream promoter and consist of an acidic N-terminal transactivation domain, a central DNA binding domain and a C-terminal oligomerization domain. The ¿N isoforms, produced from an intronic promoter, are capable of forming protein complexes with p53 family proteins to inhibit the function of TAp63 and p53 family members by sequestering or competing for target promoters it. Furthermore, the TAp63 and ¿Np63 isoforms undergo alternative splicing yielding different C-termianl tails. Unlike p53, which is dispensable for normal development, p63 is critical for the development of stratified epithelial tissues, such as epidermis, breast and prostate. p63 -/- mice completely lack stratified squamous epithelial and their derivatives, including epidermal appendages and mammary lacrimal, and salivary glands. Due to the lack of an epidermal barrier, these mice dehydrate and die shortly after birth. In addition, p63 -/- mice have major defects in limb and craniofacial development. Limb truncation in p63-/- mice are due to failure to maintain or differentiate the apical ectodermal ridge, a structure required for limb extension and dependent on coordinated epithelial-mesenchymal interactions. Several autosomal dominant inherited human syndromes have have been mapped to the p63 gene. These syndromes have various combinations of limb malformations fitting the split hand-split foot spectrum, orofacial clefting, and extodermal dusplasia. p63 may also play a role in tumorigenesis. To explore these roles, we have generated GST-fusion constructs using pGEX-2TK vector through PCRbased cloning approach. We transformed these plasmids into BL21 E.Coli strain, induced by IPTG and examined the expression levels of these fusion proteins. We are purifying the GST-p63 fusion proteins using a GST-pull down approach. The purified GST-p63 fusion proteins are being used to pull down proteins from lysates of HeLa cells. Proteins are separated on SDS-PAGE and stained . Individual protein bands are being subjected to mass spectrometry analysis. The results have allowed the identification of key proteins.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 p63 是 p53 同源物，被转录为源自差异表达的六种亚型 反式激活 (TA) 亚型类似于 p53，是通过使用上游启动子产生的，由酸性 N 端反式激活结构域、中央 DNA 结合结构域和 C 端寡聚化结构域组成。这 由内含子启动子产生的 N 亚型能够与 p53 家族蛋白形成蛋白复合物，通过隔离或竞争靶启动子来抑制 TAp63 和 p53 家族成员的功能。此外，TAp63 和 ¿ Np63 同种型经过选择性剪接，产生不同的 C 末端尾部，与正常发育所必需的 p53 不同，p63 对于分层上皮组织（如表皮、乳腺和前列腺）的发育至关重要，p63 -/- 小鼠缺乏完全分层的鳞状细胞。上皮及其衍生物，包括表皮附属物和乳腺泪腺，以及唾液腺。表皮屏障，这些小鼠出生后不久就会脱水并死亡。此外，p63-/- 小鼠的肢体和颅面发育存在重大缺陷，这是由于未能维持或分化顶端外胚层脊。肢体伸展所需的结构，依赖于协调的上皮间质相互作用，几种常染色体显性遗传的人类综合征已被映射到 p63 基因。适合裂手裂足谱、口面裂和外皮发育不良的各种肢体畸形组合也可能在肿瘤发生中发挥作用。为了探索这些作用，我们通过基于 PCR 的克隆使用 pGEX-2TK 载体生成了 GST 融合构建体。我们将这些质粒转化到 BL21 大肠杆菌菌株中，通过 IPTG 诱导并检测这些融合蛋白的表达水平。使用 GST 下拉方法纯化的 GST-p63 融合蛋白用于从 HeLa 细胞裂解物中下拉蛋白质，并对各个蛋白质条带进行染色。质谱分析的结果可以鉴定关键蛋白质。