Protein fragments as cotranslationally-acting inhibitors

项目总结 大多数现有的药物通过与表面可接触的全折叠蛋白质残基结合来发挥作用。许多占主导地位 负性多肽也以这种方式发挥作用，无活性的多肽可以防止形成 功能性低聚物。然而，在某些情况下，单体酶的片段也能够起到 显性负性，阻止其来源蛋白质的重折叠。此外，形成复合体的蛋白质 可以绑定到其交互伙伴的主动翻译的新生链条，并充当其 折叠。这些结果表明，蛋白质片段可能通常能够通过结合到 部分折叠的新生目标蛋白质链。对这种抑制剂的广泛敏感性可能会 通过将整个蛋白质序列考虑为潜在药物，使药物开发发生革命性变化 目标。我建议确定共翻译抑制片段的流行率，调查 这些片段与序列和结构特征的关系以及它们的关联性测试 在体外和体内，带有其目标蛋白的新生链条的片段。 菲尔兹实验室最近开发了一种高通量的蛋白质片段显性负活性分析方法 在活体内，根据片断耗竭的测量选择。在目标1中，我将生成一个质粒库 编码六种酵母蛋白的片段。我将在酵母细胞上进行显性阴性片段分析 在标准条件和预计会在全球范围内减慢翻译速度的条件下携带该库， 包括氨基酸限制。我还将进行显性阴性片段分析，在这些分析中，我特别 通过将目标蛋白的编码序列替换为密码子去优化的变体来减慢目标蛋白的翻译。 翻译减速应该会增加共翻译结合片段的抑制作用。在《目标2》中，我会 使用全局翻译减速条件来检测共翻译作用的蛋白质片段 全基因组使用平衡的酵母ORF片段文库。我会将推断出的片段结合位点与 通过计算预测了翻译停顿位置和球状域分配。我希望 共翻译抑制片段将被C末端富含到翻译停顿，尤其是停顿 在域之间。即使在缺乏实验数据的情况下，这些预测也将使我能够提出目标 协译作用抑制物的位置。在目标3中，我将测试有希望片段是否与 新生的目标蛋白质链。我将在体外转录和翻译目标蛋白，并检测片段 在翻译过程中存在，用于抑制靶标活性和靶标结合亲和力。我还会有选择性地表演 核糖体图谱实验中，我拉下了核糖体-新生链状复合体 结合标准的核糖体图谱实验，计算浓缩效率。 根据这些结果，我将确定每个片段的全基因组共翻译结合靶点(S)，以及 其新生的链结合位点，提供了分子水平的了解该片段的作用机制。

PROJECT SUMMARY Most existing drugs act by binding to surface-accessible residues of fully-folded proteins. Many dominant negative polypeptides function in this manner as well, with an inactive polypeptide preventing formation of a functional oligomer. However, in some cases, fragments of monomeric enzymes are also capable of acting as dominant negatives, preventing refolding of their proteins of origin. Additionally, proteins that form complexes may bind to the actively translating nascent chains of their interaction partners and act as chaperones for their folding. These results suggest that protein fragments might generally be able to act as inhibitors by binding to the partially folded nascent chains of target proteins. Broad susceptibility to such inhibitors would potentially revolutionize drug development by enabling consideration of entire protein sequences as potential drug targets. I propose to determine the prevalence of cotranslationally-inhibitory fragments, investigate the relationship of these fragments with sequence and structural features, and test for association of such fragments with nascent chains of their target proteins both in vitro and in vivo. The Fields lab recently developed a high-throughput assay for dominant negative activity of protein fragments in vivo, based on measurement of fragment depletion in a selection. In Aim 1, I will generate a plasmid library encoding fragments of six yeast proteins. I will perform the dominant negative fragment assay on yeast cells carrying this library under both standard conditions and conditions expected to globally slow translation, including amino acid limitation. I will also perform dominant negative fragment assays in which I specifically slow down translation of target proteins by replacing their coding sequences with codon-deoptimized variants. Translational slowdown should yield increased inhibition by cotranslationally-binding fragments. In Aim 2, I will use global translational slowdown conditions to assay for cotranslationally-acting protein fragments genomewide using a balanced yeast ORF fragment library. I will compare inferred fragment binding sites with computationally predicted translational pause sites and globular domain assignments. I expect cotranslationally-inhibitory fragments will be enriched C-terminal to translational pauses, especially pauses between domains. Even in the absence of experimental data, these predictions will allow me to propose target sites for cotranslationally-acting inhibitors. In Aim 3, I will test whether promising fragments associate with nascent chains of target proteins. I will transcribe and translate target proteins in vitro, and assay fragments present during translation for inhibition of target activity and target-binding affinity. I will also perform selective ribosome profiling experiments in which I pull down ribosome-nascent-chain complexes associated with fragments of interest, alongside standard ribosome profiling experiments, to calculate enrichment efficiencies. From these results, I will determine genomewide cotranslational binding target(s) of each fragment, as well as its nascent chain binding sites, providing molecular-level insight into the fragment’s mechanism of action.