权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

项目总结