Localised protein synthesis in fibroblasts during cell spreading and migration in 3D culture

3D 培养中细胞扩散和迁移过程中成纤维细胞的局部蛋白质合成

• 批准号: BB/H018956/1
• 负责人: Simon Morley
• 金额: $ 41.91万
• 依托单位: University of Sussex
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2010
• 资助国家: 英国
• 起止时间: 2010 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FH018956%2F1
• 关键词: Localised; protein; synthesis; fibroblasts; during

The ability of cells to move about is an important process in development, generation of blood vessels and repair of damaged tissues after injury, requiring new proteins to be made. For cells to be able to do this, critical information stored in the gene sequences of the genetic material (DNA) has to be decoded by the cell to produce a wide variety of essential proteins of the right type, in the right amount and at the right time. The general transfer of information from DNA to protein is carried out by the messenger RNA (mRNA), which is a copy of the DNA sequence. When required, this mRNA has to be decoded into protein in different parts of the cell by a complex, highly regulated machine termed a ribosome, in a process known as translation. Localised protein synthesis allows the cell to make the protein exactly where and when it needs it in the cell without having to waste time and energy moving the protein around to the correct location. To work efficiently, accurately, and to allow the ribosome to function in the best interests of the cell, this machinery requires helper proteins (translation initiation factors; eIF) that interact with each other, and also make sure that the mRNA and the ribosome come together into a highly regulated, large initiation complex to make the proteins required. So how does the cell make this happen in the right place at the right time? The interaction of the initiation factors themselves is a major site for regulation in mammalian cells. One protein, 4E-binding protein 1 (4E-BP1) prevents the interaction of eIF4E with the scaffold protein, eIF4G, and stops the recruitment of mRNA to the ribosome and halts protein synthesis. When protein synthesis is needed, the cell signals to 4E-BP1 to release the eIF4E/mRNA from the 4E-BP1/eIF4E/mRNA complex to let it work. The cell does this by marking the 4E-BP1, eIF4E, eIF4G and ribosomes with phosphate groups in a process known as phosphorylation. This modification promotes 4E-BP1 release from eIF4E/mRNA which can subsequently bind to eIF4G and form the multi-protein initiation complex required to make the correct types and amounts of protein needed. However, we still do not know how the cell controls localised protein synthesis in cells which are in the process of migrating. From 'looking' inside the cell with specialised microscopy techniques, we know that the initiation factors are discretely localised to specific regions in the cell; they are not just floating about. In the work described here we want to investigate where and how fibroblasts cells localise their translational machinery when they are prompted to migrate. We then want to understand which signals are required to bring about this localisation and show whether these regions reflect active areas where proteins are being made as the cell moves about in culture. These studies will substantially increase our general understanding of the significance of the control of protein synthesis in the regulation of cell growth and migration, opening up new potential avenues for controlling cancer cells which have acquired the ability to move about the body.

细胞移动的能力是发育、血管生成和损伤后受损组织修复的重要过程，需要制造新的蛋白质。为了让细胞能够做到这一点，存储在遗传物质 (DNA) 基因序列中的关键信息必须被细胞解码，以产生正确类型、正确数量的各种必需蛋白质。正确的时间。从 DNA 到蛋白质的一般信息传递是由信使 RNA (mRNA) 执行的，它是 DNA 序列的副本。当需要时，这种 mRNA 必须通过称为核糖体的复杂、高度调控的机器在细胞的不同部分解码成蛋白质，这一过程称为翻译。局部蛋白质合成使细胞能够在细胞需要的时间和地点准确地制造蛋白质，而不必浪费时间和精力将蛋白质移动到正确的位置。为了高效、准确地工作，并让核糖体按照细胞的最佳利益发挥作用，这一机制需要相互相互作用的辅助蛋白（翻译起始因子；eIF），并确保 mRNA 和核糖体相互结合。一起形成高度调控的大型起始复合物以产生所需的蛋白质。那么细胞如何在正确的时间、正确的地点实现这一点呢？起始因子本身的相互作用是哺乳动物细胞中调节的主要位点。一种蛋白质 4E 结合蛋白 1 (4E-BP1) 可阻止 eIF4E 与支架蛋白 eIF4G 的相互作用，并阻止 mRNA 募集到核糖体并停止蛋白质合成。当需要蛋白质合成时，细胞向 4E-BP1 发出信号，从 4E-BP1/eIF4E/mRNA 复合物中释放 eIF4E/mRNA，使其发挥作用。细胞通过磷酸化过程用磷酸基团标记 4E-BP1、eIF4E、eIF4G 和核糖体来实现这一点。这种修饰促进 4E-BP1 从 eIF4E/mRNA 中释放，随后可以与 eIF4G 结合并形成生成正确类型和数量的所需蛋白质所需的多蛋白起始复合物。然而，我们仍然不知道细胞如何控制迁移过程中细胞的局部蛋白质合成。通过使用专门的显微镜技术“观察”细胞内部，我们知道启动因子离散地定位于细胞中的特定区域；它们不只是漂浮着。在这里描述的工作中，我们想要研究成纤维细胞在被提示迁移时将其翻译机制定位在何处以及如何定位。然后，我们想要了解实现这种定位需要哪些信号，并显示这些区域是否反映了细胞在培养物中移动时产生蛋白质的活性区域。这些研究将大大提高我们对控制蛋白质合成在调节细胞生长和迁移中的重要性的一般理解，为控制已获得在体内移动能力的癌细胞开辟新的潜在途径。

项目成果

Murine norovirus 1 (MNV1) replication induces translational control of the host by regulating eIF4E activity during infection.

• DOI: 10.1074/jbc.m114.602649
• 发表时间: 2015-02-20
• 期刊: The Journal of biological chemistry
• 作者: Royall E;Doyle N;Abdul-Wahab A;Emmott E;Morley SJ;Goodfellow I;Roberts LO;Locker N
• 通讯作者: Locker N

Translation initiation factors and active sites of protein synthesis co-localize at the leading edge of migrating fibroblasts

翻译起始因子和蛋白质合成活性位点共定位于迁移成纤维细胞的前缘

• DOI: 10.1042/bj20110435
• 发表时间: 2011
• 期刊: Biochemical Journal
• 影响因子: 4.1
• 作者: Willett M

The helicase, DDX3X, interacts with poly(A)-binding protein 1 (PABP1) and caprin-1 at the leading edge of migrating fibroblasts and is required for efficient cell spreading.

• DOI: 10.1042/bcj20170354
• 发表时间: 2017-08-30
• 期刊: The Biochemical journal
• 作者: Copsey AC;Cooper S;Parker R;Lineham E;Lapworth C;Jallad D;Sweet S;Morley SJ
• 通讯作者: Morley SJ

Sumoylation of eIF4A2 affects stress granule formation.

• DOI: 10.1242/jcs.184614
• 发表时间: 2016-06-15
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Jongjitwimol J;Baldock RA;Morley SJ;Watts FZ
• 通讯作者: Watts FZ