Interplay of structure, activity and localization in tRNA nucleotidyltransferase function

tRNA 核苷酸转移酶功能中结构、活性和定位的相互作用

• 批准号: RGPIN-2020-04615
• 负责人: Joyce, Paul
• 金额: $ 2.33万
• 依托单位: Concordia University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=740069
• 关键词: Interplay; structure; activity; localization; tRNA

Proteins are the doers in cells. They are involved in building, degrading, transporting, importing and exporting cellular components. In order to carry out these functions, proteins must fold correctly into three-dimensional shapes that enable precise interactions with other molecules such as proteins, DNA, RNA, metabolites, metal ions, or combinations of these. In addition, proteins must function only when and where required. For example, some proteins like hemoglobin, which is packed in red blood cells and carries oxygen to our cells, must function for a long time, while other proteins, like those involved in blood clotting, must act quickly but only transiently. Moreover, our cells are not simple bags of molecules, but instead are comprised of subcellular compartments (e.g., the nucleus or mitochondrion or chloroplast) where different processes occur. So, proteins must be sorted to their proper cellular destinations to perform those activities. The proper functioning of any protein results from a combination of four factors: location, activity, structure and stability. To understand how these four factors are entwined, we study one model protein called tRNA nucleotidyltransferase (tRNA-NT). This vital protein is an enzyme that builds a small extension on the end of transfer RNAs (tRNAs) so that amino acids can be attached. These tRNAs with amino acids act as adaptor molecules to translate the genetic code into the sequence of amino acids in a protein. Therefore, without tRNA-NT cells do not make proteins. Because tRNA synthesis and repair occur in multiple places in the cell (nucleus, mitochondrion, cytosol and in plants, chloroplast) tRNA-NT must be directed to these locations. To study tRNA-NT, we first make changes in specific regions of the protein to see how these changes affect location, activity, structure and stability. Some of the changes that we have made affected multiple parameters. For example, we showed that changing certain amino acids in a plant tRNA-NT reduced that variant's thermal stability, eliminated its ability to extend tRNA (reduced activity), and caused a change in intracellular distribution (changed localization). Similarly, we showed that a single amino acid change in the yeast enzyme could reduce its thermal stability and activity. We will use a wide variety of cell and molecular biological approaches as well as biochemical and biophysical techniques to study the interplay among location, activity, structure and stability in tRNA-NT function. We also are identifying proteins that interact with tRNA-NT to ensure proper maintenance of activity and localization in cells. Our studies allow us to develop a better understanding of how these factors combine to ensure protein function. This fundamental knowledge will enable us to optimize delivery of functional proteins in cells.

蛋白质是细胞中的行动者。它们参与细胞成分的构建、降解、运输、进口和出口。为了实现这些功能，蛋白质必须正确折叠成三维形状，从而能够与其他分子（如蛋白质、DNA、RNA、代谢物、金属离子或其组合）进行精确的相互作用。此外，蛋白质必须仅在需要时和需要时发挥作用。例如，一些蛋白质，如血红蛋白，它被包装在红细胞中并将氧气运送到我们的细胞中，必须长时间发挥作用，而其他蛋白质，如那些参与血液凝固的蛋白质，必须迅速但短暂地发挥作用。此外，我们的细胞不是简单的分子袋，而是由亚细胞区室组成（例如，细胞核或叶绿体），在那里发生不同的过程。因此，蛋白质必须被分类到它们适当的细胞目的地来执行这些活动。任何蛋白质的正常功能都取决于四个因素：位置，活性，结构和稳定性。为了了解这四个因子是如何被激活的，我们研究了一种称为tRNA核苷酸转移酶（tRNA-NT）的模型蛋白。这种重要的蛋白质是一种酶，它在转移RNA（tRNA）的末端建立一个小的延伸，以便可以连接氨基酸。这些含有氨基酸的tRNA充当衔接分子，将遗传密码翻译成蛋白质中的氨基酸序列。因此，没有tRNA-NT细胞不制造蛋白质。由于tRNA的合成和修复发生在细胞的多个位置（细胞核、线粒体、胞质溶胶和植物中的叶绿体），因此tRNA-NT必须定向到这些位置。 为了研究tRNA-NT，我们首先改变蛋白质的特定区域，看看这些变化如何影响位置，活性，结构和稳定性。我们所做的一些改变影响了多个参数。例如，我们发现改变植物tRNA-NT中的某些氨基酸会降低该变体的热稳定性，消除其延伸tRNA的能力（降低活性），并导致细胞内分布的变化（改变定位）。同样，我们发现酵母酶中的单个氨基酸变化可以降低其热稳定性和活性。我们将使用各种各样的细胞和分子生物学方法以及生物化学和生物物理技术来研究tRNA-NT功能的位置，活性，结构和稳定性之间的相互作用。我们还正在鉴定与tRNA-NT相互作用的蛋白质，以确保在细胞中正确维持活性和定位。我们的研究使我们能够更好地了解这些因素如何联合收割机，以确保蛋白质的功能。这些基础知识将使我们能够优化细胞中功能蛋白的递送。