# Defining a pathway for mitochondrial heme trafficking

> **NIH NIH R01** · CLEVELAND CLINIC LERNER COM-CWRU · 2024 · $536,531

## Abstract

ABSTRACT
Hemeproteins play vital transport, enzymatic, and signaling roles that fundamentally impact our cardiovascular,
pulmonary, digestive, neurological, and immune systems. Cells must transport mitochondrial heme to proteins
that mature and function outside the mitochondria. Our goal is to understand how intracellular heme delivery
takes place and is regulated in mammals. Cytosolic heme delivery proteins have been postulated but their
identities are unclear. We found that GAPDH binds mitochondrially-generated heme and that its heme binding
is essential for delivery to eight different hemeprotein targets. We hypothesize that GAPDH-dependent heme
delivery is fundamental for hemeprotein function, and we propose to discern mechanisms, scope, and
regulation of GAPDH-dependent heme delivery. Our experiments utilize purified proteins and cell culture,
which provides a facile path to molecular-level discoveries and a robust means to validate their biological
relevance. We have ways to control cell heme production, monitor GAPDH-heme binding and transfer in live
cells and between proteins, assess heme delivery to targets, characterize GAPDH-target complexes, and
determine their relevance for heme delivery.
AIM 1. How does GAPDH participate in intracellular heme delivery? We hypothesize GAPDH may bind
directly to the target proteins to deliver heme and found it does so with at least four proteins (apo-sGCβ, IDO1,
TDO, and Mb) in cells and purified form. We will: (i) identify interface regions in each GAPDH-hemeprotein
complex using HDx-MS, MS-cleavable crosslinking, and NMR approaches; (ii) use mutagenesis to test the role
of GAPDH-target protein contacts in heme deliveries; (iii) structurally characterize the GAPDH-heme complex
and GAPDH-hemeprotein complexes by crystallography and single particle EM; (iv) utilize a GAPDH-biolD2-HA
fusion protein to biotinylate intermediate or middleman proteins that associate with GAPDH during heme
transfers; (v) deploy GAPDH surface charge mutants to independently probe GAPDH-target binding and role in
heme transfers; (vi) perform heme transfer experiments with purified GAPDH & target proteins; (vii) screen for
GAPDH-dependent heme deliveries to cell heme exporters (FLVCR1a & ABCG2), cytochrome P450’s (CYP2D6
& CYP3A4), two peroxidases (LPO & EPO), an NADPH oxidase (NOX5), and heme oxygenases 1 & 2.
AIM 2. What controls GAPDH heme acquisition & deliveries? Cell & molecular mechanisms that control
GAPDH heme acquisition and release are unknown. We will: (i) determine if mitochondrial heme exporter
FLVCR1b and/or ER heme exporter PGRMC2 is a heme source for GAPDH; (ii) test if GAPDH heme acquisition
involves direct interaction with the exporters; (iii) investigate if GAPDH-heme level in cells is regulated by cell
heme exporters FLVCR1a & ABCG2; (iv) examine if post-translational GAPDH modification, chaperone hsp90,
or azole drugs control GAPDH heme acquisition and delivery.

## Key facts

- **NIH application ID:** 10904908
- **Project number:** 5R01GM148664-02
- **Recipient organization:** CLEVELAND CLINIC LERNER COM-CWRU
- **Principal Investigator:** DENNIS J STUEHR
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $536,531
- **Award type:** 5
- **Project period:** 2023-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10904908

## Citation

> US National Institutes of Health, RePORTER application 10904908, Defining a pathway for mitochondrial heme trafficking (5R01GM148664-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10904908. Licensed CC0.

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