Protein fragments as cotranslationally-acting inhibitors

项目摘要 大多数现有的药物通过与完全折叠的蛋白质的表面可及残基结合而起作用。许多主导 负性多肽也以这种方式起作用，无活性多肽阻止 功能性低聚物然而，在某些情况下，单体酶的片段也能够充当 显性负性，阻止其蛋白质的再折叠。此外，形成复合物的蛋白质 可以与它们的相互作用伙伴的活跃翻译的新生链结合，并充当它们的伴侣。 折页.这些结果表明，蛋白质片段通常能够通过结合到 靶蛋白的部分折叠的新生链。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而彻底改变药物开发 目标的我建议确定凝聚抑制片段的流行，调查 这些片段与序列和结构特征的关系，并测试这些片段的相关性。 片段与其靶蛋白的新生链在体外和体内。 菲尔兹实验室最近开发了一种高通量检测蛋白质片段显性负活性的方法 在体内，基于在选择中片段消耗的测量。在目标1中，我将生成质粒文库， 编码六种酵母蛋白的片段。我将对酵母细胞进行显性阴性片段分析 在标准条件和预期会使整体翻译变慢的条件下携带该文库， 包括氨基酸限制。我还将进行显性阴性片段测定， 通过用密码子去优化的变体替换靶蛋白的编码序列来减缓靶蛋白的翻译。 翻译减慢应产生增加的抑制作用的coconstructionally结合片段。在目标2中，我将 使用全局翻译减慢条件来测定协同作用蛋白片段 使用平衡的酵母ORF片段文库在全基因组范围内进行。我将比较推断的片段结合位点与 计算预测的翻译暂停位点和球状结构域分配。我预计 协同抑制性片段将富集在翻译停顿，特别是停顿的C-末端， 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。 用于协同作用抑制剂的位点。在目标3中，我将测试有希望的片段是否与 新生的靶蛋白链。我将在体外转录和翻译靶蛋白，并分析片段 在翻译期间存在用于抑制靶活性和靶结合亲和力。我也会选择性地 核糖体分析实验中，我拉下核糖体新生链复合物与 感兴趣的片段，以及标准核糖体分析实验，以计算富集效率。 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，以及 它的新生链结合位点，提供分子水平的洞察片段的作用机制。

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.