# Expanding the set of genetically encoded tools for compartment-specific manipulation of redox metabolism in living cells

> **NIH NIH R35** · SCINTILLON INSTITUTE FOR PHOTOBIOLOGY · 2022 · $246,546

## Abstract

Abstract
The metabolic environment that cells face has profound effects on cellular behavior. This is especially true for
the reduction-oxidation (redox) environment, but many aspects of how redox metabolism is regulated and how
it directs cellular decisions are poorly understood. In order to systematically address these pressing questions,
it is necessary to have tools with which key contributors to the cellular redox environment can be safely and
directly modulated with spatial and, most importantly, temporal resolution. We previously used a H2O-forming
NADH oxidase from Lactobacillus brevis (LbNOX) to decrease the NADH/NAD+ ratio when ectopically
expressed in cytoplasm or mitochondria of mammalian cells. Furthermore, we engineered a variant of this
enzyme with strict specificity towards NADPH (TPNOX). We subsequently employed both LbNOX and
TPNOX as genetically encoded tools to show that NAD+ regeneration but not ATP production is a critical
requirement of proliferation of mammalian cells. In our original MIRA ESI application, we plan to continue
development of evolution-inspired, genetically encoded tools for spatiotemporal modulation of key cellular
redox parameters. In Project 1, we plan to expand our toolkit by developing a genetically encoded tool for the
direct modulation of NADH reductive stress (i.e. increased NADH/NAD+ ratio). In Project 2, we will elucidate
the metabolic and cellular consequences of the NADH reductive stress in various backgrounds. We will use
Drosophila flies to directly test whether redox modulation in either the oxidative or reductive direction is correlated
with stress resistance, healthspan and lifespan. In Project 3 we will combine protein engineering and imaging
techniques to develop versions of our tools where the corresponding enzymatic activity is controlled by small
molecule or light stimulation to achieve temporal control of the corresponding redox pairs. Using our tools,
we will also illuminate the role of various redox active small molecules, including systemic mitochondrial
complex I inhibition and associated redox imbalance, in the progression of neuronal loss in Parkinson’s disease
(PD). This Administrative Supplement requests the acquisition of a BioTek Cytation C10 confocal imaging
reader, which would allow us to use automated microscopy to quantify multiple cell parameters simultaneously,
including cellular size and shape, morphological and functional changes in subcellular structures, inter-organelle
communication and to image fluorescence-based biosensors. In summary, access to a BioTek Cytation C10
instrument will significantly accelerate experiments described in Projects 1-3.

## Key facts

- **NIH application ID:** 10582469
- **Project number:** 3R35GM142495-02S1
- **Recipient organization:** SCINTILLON INSTITUTE FOR PHOTOBIOLOGY
- **Principal Investigator:** Valentin Cracan
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $246,546
- **Award type:** 3
- **Project period:** 2021-07-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10582469

## Citation

> US National Institutes of Health, RePORTER application 10582469, Expanding the set of genetically encoded tools for compartment-specific manipulation of redox metabolism in living cells (3R35GM142495-02S1). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10582469. Licensed CC0.

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