# Small proteins and the regulation of protein degradation

> **NIH NIH FI2** · U.S. NATIONAL INST/CHILD HLTH/HUMAN DEV · 2022 · —

## Abstract

ABSTRACT
The characterization of small proteins (<50 amino acids) is an emerging and underexplored field of study.
Small proteins are generally thought to be induced by stress and regulate the response to that stress.
However, they can also be toxic when overexpressed. Therefore, their levels must be tightly regulated both
transcriptionally and posttranslationally. Work in Escherichia coli and Salmonella enterica serovar Typhimurium
has revealed small proteins required for growth when magnesium (Mg2+) is limiting. Several of these proteins
have been shown to affect the FtsH-dependent degradation of transporters required for the uptake of Mg2+.
The molecular details of how small proteins influence the stability of the Mg2+ transporters they interact with
remains unknown. Here we aim to understand in molecular detail, using single-particle cryo-electron
microscopy (Cryo-EM), how the E. coli small protein MgtS binds the P-type ATPase magnesium transporter
MgtA. We hypothesize that the MgtA and MgtS binding interface overlaps with the protease recognition
sequence (degron) thereby masking its recognition. To test this hypothesis, structural and in silico models will
be used to make mutations that disrupt the predicted binding interface of MgtA and MgtS, and their stabilities
will be monitored both in vitro and in vivo. Additionally, we will carry out pulsed stable isotope labeling of amino
acids in cell culture (pSILAC) with mass spectrometry-based proteomics during the response and recovery
from magnesium limitation or other stress conditions to monitor small protein degradation. Top candidates from
our global survey of small protein stability will be tagged and their degradation tested. Co-immunoprecipitation
(co-IP) and mass spectrometry will identify potential interacting partners. Finally, we will test which proteases
degrade our candidate small proteins and perform degradation assays to observe how the presence or
absence of the small protein influences the larger protein’s stability. The results from this proposal will provide
critical insights into how small proteins regulate the stability of the larger proteins they bind and identify novel
small protein protease adaptors. Given that small proteins are found in the gut microbiota, bacterial pathogens,
and in eukaryotes, our approach will serve as a model for studying small protein-big protein interaction and
small protein turnover in other model organisms.

## Key facts

- **NIH application ID:** 10498062
- **Project number:** 1FI2GM146628-01
- **Recipient organization:** U.S. NATIONAL INST/CHILD HLTH/HUMAN DEV
- **Principal Investigator:** Rilee Zeinert
- **Activity code:** FI2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** —
- **Award type:** 1
- **Project period:** 2022-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10498062

## Citation

> US National Institutes of Health, RePORTER application 10498062, Small proteins and the regulation of protein degradation (1FI2GM146628-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10498062. Licensed CC0.

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