# Protein fragments as cotranslationally-acting inhibitors

> **NIH NIH F32** · UNIVERSITY OF WASHINGTON · 2021 · $27,315

## Abstract

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...

## Key facts

- **NIH application ID:** 10134797
- **Project number:** 5F32GM134557-02
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Andrew Savinov
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $27,315
- **Award type:** 5
- **Project period:** 2020-04-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10134797

## Citation

> US National Institutes of Health, RePORTER application 10134797, Protein fragments as cotranslationally-acting inhibitors (5F32GM134557-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10134797. Licensed CC0.

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