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; 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.

该副本是使用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这是调查员的机构。 p63是一种p53同源物，被转录为从差异衍生的六种同工型 启动子使用和替代剪接。类似于p53的反式激活（TA）同工型通过使用上游启动子而产生，由酸性N末端反式反式激活结构域，中央DNA结合结构域和C末端寡聚域组成。由内含子启动子产生的N同工型能够与p53家族蛋白形成蛋白质复合物，以隔离或竞争目标启动子IT，以抑制TAP63和p53家族成员的功能。此外，TAP63和„ NP63同工型经历替代剪接，产生不同的C-术语尾巴。与正常发育的p53不同，p63对于表皮，乳腺和前列腺等分层上皮组织的发展至关重要。 p63 - / - 小鼠完全缺乏分层的鳞状上皮及其衍生物，包括表皮附属物和乳腺乳酸和唾液网格。由于缺乏表皮屏障，这些小鼠出生后不久死亡。此外，p63 - / - 小鼠在肢体和颅面发育方面存在主要缺陷。 p63 - / - 小鼠中的肢体截断是由于未能维持或区分根尖外胚层脊，这是肢体延伸所需的结构，并取决于协调的上皮 - 间质相互作用。几种常染色体显性遗传性人类综合征已映射到p63基因。这些综合征具有各种肢体畸形的组合，适合分裂的脚谱，口面裂和硬皮增生。 p63也可能在肿瘤发生中起作用。为了探索这些角色，我们通过基于PCRB的克隆方法使用PGEX-2TK矢量生成了GST融合构建体。我们将这些质粒转化为由IPTG诱导的BL21大肠杆菌菌株，并检查了这些融合蛋白的表达水平。我们正在使用GST-PULL降低方法纯化GST-P63融合蛋白。纯化的GST-P63融合蛋白用于从HeLa细胞裂解物中拉下蛋白质。蛋白质在SDS-PAGE上分离并染色。各个蛋白质条带正在进行质谱分析。结果允许鉴定关键蛋白质。