# Revealing the essential functions of mitochondrial NADPH and NADK2 for cell growth and proliferation

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2022 · $328,000

## Abstract

SUMMARY: To grow and proliferate, cells need to fulfill three key metabolic demands: increased biosynthesis,
sufficient energy supply, and maintenance of redox homeostasis. The latter demand is particularly important for
sustained growth, because increased rate of cellular metabolic activity during cell proliferation results in elevated
levels of reactive oxygen species (ROS), which can have detrimental effects on cell growth. Nicotinamide
adenine dinucleotide phosphate (NADPH) is a principal supplier of reducing power for biosynthesis of
macromolecules and protection against oxidative stress. The total cellular pool of NADPH is regulated by the
activity of NAD kinases (NADK), enzymes that catalyze the phosphorylation of NAD+ to NADP+, the rate-limiting
substrate for NADPH production. Mammalian cells express two NAD kinases, cytosolic NADK, and mitochondrial
NADK2, which generate compartment-specific reducing power. Recently, we discovered that the activity of
NADK is stimulated by the phosphoinositide 3-kinase (PI3K) - Akt pathway to boost the NADP(H) production for
cell growth, but the importance of NADK2 and the overall role of mitochondrial NADPH in cell growth has yet to
be established. Mutations in NADK2 have been observed in patients with various neurological and
developmental disorders. Therefore, defining the key functions of NADK2 and mitochondrial NADP(H) is critical
and relevant to human health. This proposal builds on our finding that decreasing mitochondrial NADP(H) levels
through depletion of NADK2 renders cells uniquely proline auxotroph. Cells with NADK2 deletion fail to
synthesize proline and rely on exogenous proline for their growth. Proline is critical for protein synthesis, and,
unexpectedly, for nucleotide synthesis, in NADK2-deficient cells. We propose three Specific Aims to establish
the functions of NADK2 and mitochondrial NADP(H) that are essential for cell growth and proliferation and
relevant for proliferative diseases and NADK2 deficiency in humans. In Aim 1, we propose to define the molecular
mechanisms by which NADK2 and mitochondrial NADPH support proline biosynthesis and evaluate the effects
of NADK2 patient mutations on proline synthesis. In Aim 2, we will determine the mechanisms of how NADK2
deficiency and reduced proline abundance affect flux through the de novo and salvage nucleotide synthesis
pathways. In Aim 3, we will assess the requirement of NADK2 and mitochondrial NADPH for tumor growth and
evaluate the therapeutic potential of targeting NADK2 in combination with dietary restriction of proline. This
proposal will establish the primary functions of mitochondrial NADP(H) and NADK2 that are essential for cell
growth and proliferation, thereby informing us on new therapeutic strategies to combat proliferative diseases and
NADK2 deficiency in humans.

## Key facts

- **NIH application ID:** 10487573
- **Project number:** 5R01GM143236-02
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Gerta N/A Hoxhaj
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $328,000
- **Award type:** 5
- **Project period:** 2021-09-15 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10487573

## Citation

> US National Institutes of Health, RePORTER application 10487573, Revealing the essential functions of mitochondrial NADPH and NADK2 for cell growth and proliferation (5R01GM143236-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10487573. Licensed CC0.

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