The structural basis of transcription factor 3C recruitment by N-myc

作为癌细胞特征的不受控制的增殖是由蛋白质的过表达或异常调节驱动的，所述蛋白质的正常生理作用是响应于生长信号驱动细胞生长。一种这样的蛋白质是转录因子N-myc，它是三种密切相关的蛋白质（c-myc、N-myc和L-myc）家族的一员，所有这些蛋白质都改变许多基因的表达。myc蛋白的过表达导致细胞增殖水平增加，并且是癌细胞的常见特征。Myc诱导衰老细胞重新进入细胞周期，颠覆细胞周期中的关键检查点，并促进细胞代谢的重新布线，以有利于细胞周期进展和生物量积累。最近发现N-myc通过与一般转录因子复合物TFIIIC相互作用来增加基因表达。这些分子共同作用，可以激活数百种基因的产生。这组被激活的基因参与了已知驱动癌症的N-myc的功能;功能如细胞周期进程，DNA复制，核苷酸代谢和端粒生物学。然而，目前尚不清楚N-myc和TFIIIC复合物如何相互识别，以及它们的相互作用如何影响它们相互作用的许多其他蛋白质。我们的研究计划的第一个目标是在蛋白质中单个氨基酸的水平上阐明N-myc：TFIIIC相互作用的分子基础。这将使用X射线晶体学、低温电子显微镜和交联质谱的正交方法来完成。第二个目的是通过在结合界面处使用突变来验证体外和细胞中的结构。最终的目的是利用这些突变开始了解其他哪些蛋白质形成N-myc：TFIIIC复合物的一部分，并开始了解N-myc与TFIIIC相互作用的机制，而不是其他已知结合相同或附近myc序列的分子。这项研究的一个潜在好处将是帮助验证myc的转录激活作为癌症药物发现的靶点。针对Myc的药物发现已经被证明是非常困难的，我们相信这个问题可以通过靶向支撑Myc致癌活性的蛋白质-蛋白质相互作用来解决。然而，尽管经过多年的研究，仍不清楚这些是什么。在这项工作中验证的突变可以由我们和其他人确定破坏N-myc：TFIIC相互作用对癌细胞增殖、检查点完整性和癌细胞代谢的影响。如果这种相互作用的破坏对癌细胞增殖有显着影响，则验证了这种相互作用作为开发靶向Myc的新疗法的潜在方法，这将是该领域的重大发展。此外，我们研究的另一个好处是，这项工作产生的结构和结构知识将可作为设计抑制剂的模板。

The uncontrolled proliferation that is characteristic of cancer cells is driven by the overexpression or aberrant regulation of proteins whose normal physiological role is to drive cellular growth in response to growth signals. One such protein is the transcription factor N-myc, one member of a family of three closely related proteins (c-myc, N-myc, and L-myc) all of which alter the expression of many genes. Overexpression of myc proteins leads to increased levels of cellular proliferation and is a frequent feature of cancer cells. Myc induces senescent cells to re-enter the cell cycle, subverts key checkpoints in the cell cycle, and facilitates the rewiring of cellular metabolism to favour both cell cycle progression and biomass accumulation. N-myc was recently found to increase gene expression via an interaction with a general transcription factor complex, TFIIIC. These molecules acting together were found to activate the production of hundreds of genes. This activated set of genes disproportionally involved in functions of N-myc known to drive cancer; functions such as cell cycle progression, DNA replication, nucleotide metabolism, and telomere biology. However, it is currently unknown how N-myc and TFIIIC complex recognise each other, and how their interaction impacts on the many other proteins they interact with. The first aim of our research proposal is to characterise the molecular basis of the N-myc:TFIIIC interaction at the level of the individual amino acids in the proteins. This will be done using orthogonal methods of X-ray crystallography, cryo-electron microscopy, and cross-linking mass spectrometry. The second aim is to validate the structure in vitro and in cells by using mutations at the binding interface. The final aim is to use these mutations to begin to understand what other proteins form part of the N-myc:TFIIIC complex and to begin to understand the mechanism by which N-myc interacts with TFIIIC over other molecules which are known to bind the same, or nearby, myc sequences. A potential benefit of this research will be to help validate transcriptional activation of myc as a target for cancer drug discovery. Drug discovery against Myc has proven very difficult and we believe the problem can be solved by targeted the protein-protein interactions that underpin Myc's oncogenic activity. However, it is not clear which these are, despite many years of research. Mutations which are validated in this work can be by us and others to determine the effect of disrupting the N-myc:TFIIC interaction on cancer cell proliferation, checkpoint integrity and cancer cell metabolism. If disruption of this interaction has significant effect on cancer cell proliferation it validates this interaction as a potential approach to develop new therapeutics that target Myc and this would be a big development in the field. Moreover, an additional benefit of our research is that structures and structural knowledge generated by this work will be available as a template for the design of inhibitors.