# Functional Dissection of Metabolic-Sensing Proline Hydroxylation Pathways

> **NIH NIH R35** · UNIVERSITY OF MINNESOTA · 2020 · $368,173

## Abstract

Project Summary
Mounting evidence have demonstrated proline hydroxylation (Hyp) as a fundamental posttranslational
modification that are highly responsive to the changes in cellular metabolic environment. During cancer
development, rapid proliferation of cancer cells in solid tumors suffers from limited oxygen supply. The hypoxia
microenvironment prevents its hydroxyproline-dependent degradation of HIFα proteins and activates hypoxia-
response cellular pathways that promote cancer cell survival in hypoxia. In addition to oxygen, the regulatory
enzyme prolyl hydroxylases are also sensitive to the concentration of iron and key mitochondria metabolites
including succinate, fumarate and alpha-ketoglutarate, making the pathway a critical metabolic sensor in cells.
Extensive studies have demonstrated that proline hydroxylation of substrate proteins regulates protein-protein
interactions or substrate protein degradation. Despite of its important roles in cell physiology and success in
targeted analysis of individual substrates, system-wide characterization and functional quantification of the
pathway have been hindered by the lack effective tools and strategies for global site-specific identification of
proline hydroxylation targets. Our overall hypothesis and long-term goal is that systematic characterization of
“proline hydroxylome” through the development of functional proteomics approaches will lead to mechanistic
understanding of the novel Hyp-mediated metabolic regulations in development and diseases. To achieve this
goal, we have developed and applied an immunoprecipitation-assisted strategy for global identification of
proline hydroxylation targets. With this strategy, we will tackle the challenge of systematic discovery and
quantification of proline hydroxylation proteome. We will develop new quantitative proteomics workflows and
apply the strategies for the identification and validation of endogenous prolyl hydroxylase targets. Using
temporal dynamics analysis, we will also reveal the target proteins that are subject to Hyp-dependent protein
degradation. Integrated data analysis will reveal the regulatory enzyme of the novel Hyp substrates and
therefore enable confident validation as well as functional characterization. In addition to the target-specific
degradation, our preliminary proteomics analysis showed that proline hydroxylation may regulate global protein
homeostasis through the regulation of proteasome activities. We will develop endogenous model systems and
novel quantitative mass spectrometry technology to determine the functional significance of proline
hydroxylation on proteasome subunits and how such regulation affect global protein homeostasis. Overall, we
anticipate that the development and application of functional proteomics technology for system-wide analysis
of proline hydroxylation targets will reveal novel metabolic-sensing pathways and potentially lead to paradigm-
shifting concepts in the fields of cancer, metabolic dis...

## Key facts

- **NIH application ID:** 9999615
- **Project number:** 5R35GM124896-04
- **Recipient organization:** UNIVERSITY OF MINNESOTA
- **Principal Investigator:** Yue Chen
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $368,173
- **Award type:** 5
- **Project period:** 2017-09-01 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9999615

## Citation

> US National Institutes of Health, RePORTER application 9999615, Functional Dissection of Metabolic-Sensing Proline Hydroxylation Pathways (5R35GM124896-04). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9999615. Licensed CC0.

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