Molecular Characterization of the Sodium/lodide Symporter (NIS)

钠/碘同向转运体 (NIS) 的分子表征

• 批准号: 7990162
• 负责人: Nancy Carrasco
• 金额: $ 10万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-03 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990162
• 关键词: Address; Affect; Amino Acids; Apical; Biogenesis; Breast; Carrier Proteins; Cell membrane; Cells; Chimera organism; Complementary DNA; Coupling; Cretinism; Defect; Degradation Pathway; Dependence; Diagnosis; Dimerization; Escherichia coli; Exhibits; Funding; Gastric mucosa; Gene Transfer; Genetic Transcription; Genomics; Human; Lateral; Malignant neoplasm of thyroid; Mediating; Medical; Membrane; Membrane Transport Proteins; Molecular; Molecular Analysis; Mutation; Neoplasm Metastasis; Oxidases; Pathway interactions; Physiological; Plasma; Play; Post-Translational Protein Processing; Process; Proteins; Regulation; Role; SLC5A5 gene; Salivary Glands; Signal Transduction; Sodium; Sterile coverings; Structure; Testing; Therapeutic; Thyroid Diseases; Thyroid Gland; Tissues; basolateral membrane; cancer therapy; clinically relevant; diagnosis evaluation; disulfide bond; insight; malignant breast neoplasm; oxidation; reconstitution; research study; symporter; trafficking

DESCRIPTION (provided by applicant): The broad objective of this project is the detailed molecular characterization of the Na+/l- symporter (NIS), a key intrinsic plasma membrane transport protein that mediates active translocation of I- into the thyroid. NIS plays a crucial role in thyroid hormogenesis and in the diagnosis and treatment of thyroid diseases, such as the use of radioiodide therapy in thyroid cancer. Starting with the isolation of the NIS cDNA earlier in this project, much progress has been made in the characterization of the protein at all levels, including the identification of functionally important residues through the characterization of NIS mutations that cause I- transport defect. NIS also mediates I- transport in other tissues, including lactating breast and breast cancer metastases, so NIS is of potential therapeutic significance in breast cancer. By means of gene transfer, NIS may also be valuable in the treatment of cancer in other tissues that do not express NIS endogenously. The studies proposed here emphasize the key process of trafficking of NIS to and from the plasma membrane, the elucidation of which is of major basic and medical relevance. The following specific aims are proposed: 1. To elucidate the molecular mechanism by which I- regulates its own NIS-mediated transport: a) does I- at high concentrations affect NIS degradation? b) what is the NIS degradation pathway? c) what is the pathway of I- -induced NIS internalization? d) does I- -induced NIS dimerization occur by intermolecular disulfide bond formation? e) is the thyroid Duox oxidase involved in the inhibition of NIS-mediated I- transport by I-? 2. To identify the determinants for polarized trafficking of NIS to the basolateral plasma membrane: a) what are the signals within the NIS Ct that target the protein to the basolateral membrane? b) can the determinants of NIS basolateral targeting reverse the targeting of the sodium/monocarboxylate transporter (SMCT) from apical to basolateral? c) is the PDZ-domain-containing protein hScrib involved in NIS targeting to and/or retention at the plasma membrane? 3. To establish new structure/function relations in NIS: we will a) ascertain the role played by amino acid residues present in transmembrane segment (TMS) IX in Na+ dependence; b) perform functional analyses of chimera NIS/SMCT proteins to assess the role of functionally important regions and/or amino acid residues in the Na+ and I- translocation pathways, as well as in Na+/l- coupling; c) undertake the solubilization, reconstitution, and purification of NIS expressed in E. coli.

描述（由申请人提供）：该项目的广泛目的是Na+/L- Somporter（NIS）的详细分子表征，这是一种关键的固有质膜转运蛋白，可介导I-的活性转运到甲状腺。 NIS在甲状腺激发以及甲状腺疾病的诊断和治疗中起着至关重要的作用，例如在甲状腺癌中使用放射性碘治疗。从该项目早期的NIS cDNA分离开始，在各个级别的蛋白质表征中取得了许多进展，包括通过表征导致I-转运缺陷的NIS突变来识别功能重要的残基。 NIS还介导其他组织中的I传输，包括乳腺癌和乳腺癌转移，因此NIS在乳腺癌中具有潜在的治疗意义。通过基因转移，NIS在其他不表达NIS的组织中的癌症治疗中也可能很有价值。此处提出的研究强调了NIS往返质膜的关键过程，该过程的阐明是主要的基础和医学相关性。提出了以下具体目的：1。阐明I-通过该分子机制调节其自身的NIS介导的运输：a）i-在高浓度下是否会影响NIS降解？ b）什么是NIS退化途径？ c）i-诱导的NIS内部化的途径是什么？ d）i-诱导的NIS二聚化是否通过分子间二硫键形成发生？ e）甲状腺氧化酶是否参与I-抑制NIS介导的I-转运的抑制？ 2。确定NIS极化向基底外侧质膜的决定因素：a）NIS CT中靶向蛋白质的信号是什么？ b）NIS基底外侧靶向的决定因素是否可以逆转钠/单羧酸盐转运蛋白（SMCT）的靶向。 c）在质膜上靶向和/或保留的NIS靶向和/或保留是否涉及含PDZ域的蛋白质？ 3。在NIS中建立新的结构/功能关系：我们将a）确定跨膜段（TMS）IX中存在的氨基酸残基在Na+依赖性中所起的作用； b）对嵌合NIS/SMCT蛋白进行功能分析，以评估Na+和I-易位途径中功能重要区域和/或氨基酸残基的作用，以及Na+/L-偶联； c）进行大肠杆菌中表达的NIS的溶解，重构和纯化。