# Deciphering human signaling networks through synthetic activation of proteins in genomically recoded organisms with multiple open codons

> **NIH NIH R01** · YALE UNIVERSITY · 2023 · $346,679

## Abstract

Project Summary
Healthy and diseased physiological states are governed by a complex web of interacting proteins that confer the
collective behavior observed in cells. These protein networks are decorated with posttranslational modifications
(PTMs) that determine their structure, function, and impart specificity for cellular signaling. Phosphorylation and
acetylation represent two common PTMs that dictate healthy and disease states in human cells. For instance,
14-3-3 proteins scaffold thousands of important phosphoproteins with evidence suggesting that acetylation can
modify its function. Current progress toward the elucidation of PTM-mediated signaling networks is hampered
by the challenge of studying transient PTMs in cells and limited methods to produce proteins containing specific
combinations of modified amino acids. Our previous efforts utilized a recoded E. coli strain (i.e., genomically
recoded organism) to synthesize all human phosphoserine proteins using a genetic code expansion technique.
We expanded this work through the development of a two hybrid like technology, named HI-P. HI-P validated
previously observed phosphorylation dependent protein-protein interactions and identified scores of novel
phosphoserine-mediated interactions across the human proteome that have been validated in biochemical- and
cell-based assays. Our approach allows for synthetic DNA inputs to direct ribosome-based phosphoprotein
synthesis and thus creates a programmable genetic tool to study the human phosphoproteome at the molecular
level. Since deciphering complex protein networks require studying the impact of multiple PTMs in isolation and
in combination, the key contribution of the proposed research is expected to expand the ability to genetically
encode phosphoserine and acetylation at precise positions in 14-3-3 proteins to reveal PTM-mediated protein-
protein interactions. Specific Aims: In this proposal, we seek to leverage a strong foundation of technologies,
expertise, and preliminary data to construct a recoded E. coli with a single stop codon (two open codons) (Aim
1), develop a protein synthesis system capable of simultaneous encoding of phosphorylated and acetylated
amino acids into proteins (Aim 2), and employ these capabilities to deconvolute PTM-mediated 14-3-3 protein
network interactions (Aim 3). Significance: This work will be significant because it will enable the expression of
programmable human proteins containing two PTMs thereby establishing a new approach to decipher complex
human signaling networks at the molecular level. We anticipate this work will elucidate novel 14-3-3 protein
network interactions governed by PTMs and enable new research into biomolecular and protein mechanisms
that can be used to develop new therapies for human disease.

## Key facts

- **NIH application ID:** 10592390
- **Project number:** 5R01GM117230-08
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Farren J. Isaacs
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $346,679
- **Award type:** 5
- **Project period:** 2015-09-25 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10592390

## Citation

> US National Institutes of Health, RePORTER application 10592390, Deciphering human signaling networks through synthetic activation of proteins in genomically recoded organisms with multiple open codons (5R01GM117230-08). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10592390. Licensed CC0.

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