Protein fragments as cotranslationally-acting inhibitors

作为共翻译作用抑制剂的蛋白质片段

基本信息

批准号：
10575062
负责人：
Andrew Savinov
金额：
$ 3.49万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2022-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10575062
关键词：
Affinity Amino Acid Sequence Amino Acids Binding Binding Sites Biological Assay C-terminal Cells Code Codon Nucleotides Complex Cycloheximide DHFR gene Data Development Dominant-Negative Mutation Drug Targeting Enzymes Escherichia coli Gene Expression Regulation Genes Genetic Transcription Growth High-Throughput Nucleotide Sequencing In Vitro Individual Libraries Measurement Measures Molecular Molecular Chaperones Open Reading Frames Peptides Pharmaceutical Preparations Phase Plasmids Predisposition Prevalence Property Protein Fragment Protein Inhibition Protein Region Proteins Ribosomes Set protein Signal Transduction Site Surface System Temperature Testing Translating Translations Variant Work Yeasts base cold temperature computerized tools drug development experimental study genome wide screen genome-wide high throughput screening in vitro Assay in vivo inhibitor insight interest member mutant novel novel strategies peptide drug polypeptide prevent protein complex protein folding ribosome profiling screening yeast genome yeast protein

项目摘要

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.

项目摘要大多数现有药物通过与完全折叠蛋白的表面访问残留物结合来起作用。许多主导阴性多肽也以这种方式发挥作用，而无效多肽则可以防止形成功能性低聚物。但是，在某些情况下，单体酶的碎片也能够充当占主导地位，防止其起源蛋白质重新折叠。另外，形成复合物的蛋白质可以与积极翻译其相互作用伙伴的新生链结合，并充当其伴侣折叠式的。这些结果表明，蛋白质片段通常可以通过结合到靶蛋白的部分折叠链。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视为潜在药物来彻底改变药物开发目标。我建议确定共转流抑制片段的患病率，研究这些片段与序列和结构特征的关系，并测试这种关联具有其靶蛋白在体外和体内的靶蛋白新生链的碎片。 Fields Lab最近开发了用于蛋白质片段主要负活性的高通量测定法在体内，基于选择中碎片耗竭的测量。在AIM 1中，我将生成一个质粒库编码六种酵母蛋白的碎片。我将在酵母细胞上执行主要的负面片段测定在标准条件和预期全球速度翻译的条件下携带此库，包括氨基酸的限制。我还将执行主要的负面片段测定法通过用密码子占优化的变体代替其编码序列来减慢目标蛋白的翻译。转化放缓应增加偶联结合片段的抑制作用。在AIM 2中，我会使用全局翻译放缓条件来分析共转倾性蛋白质片段全基因组使用平衡的酵母ORF碎片库。我将将推断的片段绑定位点与计算预测的翻译暂停站点和全局域分配。我希望共转流抑制的片段将富集C末端到翻译的停顿，尤其是停顿在域之间。即使没有实验数据，这些预测也将使我提出目标旋转作用抑制剂的位点。在AIM 3中，我将测试有希望的片段是否与靶蛋白的新生链。我将在体外转录和翻译靶蛋白，并测定片段在翻译过程中存在以抑制目标活性和目标结合亲和力。我也会执行选择性核糖体分析实验，其中我拉下了与感兴趣的片段以及标准的核糖体分析实验，以计算富集效率。从这些结果中，我将确定每个片段的全基因组共透明结合靶标，以及它的新生链结合位点，为碎片的作用机理提供了分子水平的见解。