Functional analysis of SBP2 and selenocysteine incorporation

SBP2 和硒代半胱氨酸掺入的功能分析

• 批准号: 10334457
• 负责人: PAUL R COPELAND
• 金额: $ 37.37万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-06 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10334457
• 关键词: 3' Untranslated Regions; Amino Acids; Appearance; Binding; Binding Proteins; Biological; Biological Models; Biology; Blood Vessels; Carrier Proteins; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Conserved Sequence; DNA Insertion Elements; Data; Defect; Development; Dietary Selenium; Disease; Elongation Factor; Embryo; Event; Fishes; Genes; Goals; Health; Heart Diseases; Homeostasis; Human; Hypothyroidism; IS Elements; Immune System Diseases; In Vitro; Individual; Knock-out; Life; Male Infertility; Malignant Neoplasms; Mammalian Cell; Messenger RNA; Metabolism; Molecular; Oxidation-Reduction; Oxidative Stress; Plants; Production; Proteins; Public Health; RNA-Binding Proteins; Reagent; Ribosomes; Role; Selenium; Selenocysteine; Sequence Alignment; Signal Transduction; Structure; System; Testing; Thyroid Hormones; Trace Elements; Translations; Vertebrates; Work; X-Ray Crystallography; Zebrafish; base; cancer prevention; in vivo; male fertility; novel; protein complex; protein folding; reconstitution; selenoprotein; structural biology; transcriptomics

Selenium is an essential trace element that is incorporated into 25 human proteins as the amino acid selenocysteine (Sec). The proteins that contain Sec (selenoproteins) are essential for many cellular functions including combatting oxidative stress, thyroid hormone production and protein folding. Sec is incorporated at specific UGA codons that would otherwise signal translation termination. A specialized set of factors are known to be required for Sec incorporation: a specialized elongation factor that delivers the Sec-tRNASec to the ribosome and unique RNA binding proteins that bind to a Sec insertion sequence (SECIS) in selenoprotein mRNA 3' UTRs. This SECIS-protein complex signals the ribosome to incorporate Sec instead of translation termination. Our prior work has provided molecular characterization of each of the required factors, but the mechanism by which they interact with each other and other cellular components to allow Sec incorporation remains unknown. In addition, we provide preliminary evidence that the processive incorporation of 10 Sec residues into the selenium transport protein Selenoprotein P (SELENOP) requires a unique mechanism and additional factors. The overall goals for this proposal are to determine the mechanism by which SECIS binding proteins promote single and multiple Sec incorporation events. All vertebrates possess two SECIS binding proteins encoded by separate genes: SECISBP2 (SBP2) and SECISBP2L. While the mechanism of action for SBP2 is coming into focus, the role for SECISBP2L in Sec incorporation has not been deciphered. Our preliminary data shows that SECISBP2L is essential for the processive incorporation of Sec into Selenoprotein P. As such, we have established three model systems to study the synthesis of SELENOP: in vitro translation, expression in transfected mammalian cells and a zebrafish system that will allow unprecedented access to the role of selenoprotein function during development. These are also leveraged and combined with structural biology and transcriptomics to determine how synthesis of the entire selenoproteome is regulated by SECIS binding proteins. In this proposal we propose to 1) Decipher the mechanism by which SECIS elements and SECIS binding proteins enable processive Sec incorporation into the selenium transport protein, SELENOP; 2) Utilize a zebrafish model system to determine the function of SECISBP2L and the mechanism of SELENOP synthesis in vivo; 3) Determine the molecular basis for differential selenoprotein expression. The successful completion of these aims will bring us significantly closer to our long term goal of developing reagents that will permit selective activation or inhibition of selenoprotein synthesis in vivo.

硒是一种必需的微量元素，以硒代半胱氨酸（Sec）的形式存在于25种人体蛋白质中。含有Sec（硒蛋白）的蛋白质对许多细胞功能至关重要，包括对抗氧化应激，甲状腺激素产生和蛋白质折叠。Sec在特定的UGA密码子处并入，否则其将发出翻译终止的信号。已知Sec掺入需要一组专门的因子：将Sec-tRNASec递送至核糖体的专门的延伸因子和结合硒蛋白mRNA 3'UTR中的Sec插入序列（SECIS）的独特RNA结合蛋白。该SECIS-蛋白质复合物向核糖体发出信号以并入Sec而不是翻译终止。我们先前的工作提供了每个所需因子的分子表征，但它们相互作用和其他细胞组分允许Sec掺入的机制仍然未知。此外，我们提供的初步证据表明，10秒残基的过程纳入硒转运蛋白硒蛋白P（SELENOP）需要一个独特的机制和额外的因素。该提案的总体目标是确定SECIS结合蛋白促进单个和多个Sec掺入事件的机制。所有脊椎动物都有两种由不同基因编码的SECIS结合蛋白：SECISBP 2（SBP 2）和SECISBP 2L。虽然SBP 2的作用机制正在成为焦点，但SECISBP 2L在Sec掺入中的作用尚未被破译。我们的初步数据表明，SECISBP 2L是必不可少的过程中纳入的SEC硒蛋白P。因此，我们已经建立了三个模型系统来研究合成的SELENOP：在体外翻译，表达转染的哺乳动物细胞和斑马鱼系统，将允许前所未有的访问硒蛋白功能的作用在发展过程中。这些也被利用，并与结构生物学和转录组学相结合，以确定整个硒蛋白质组的合成是如何调节SECIS结合蛋白。在这个提议中，我们建议1）破译SECIS元件和SECIS结合蛋白使Sec参与硒转运蛋白，SELENP的机制; 2）利用斑马鱼模型系统来确定SECISBP 2L的功能和体内SELENP合成的机制; 3）确定差异硒蛋白表达的分子基础。这些目标的成功完成将使我们更接近我们的长期目标，即开发允许选择性激活或抑制体内硒蛋白合成的试剂。