# Using rebuilt AAA+ enzymes to uncover the mechanisms of proteolysis at the mitochondrial inner membrane

> **NIH NIH R01** · STATE UNIVERSITY NEW YORK STONY BROOK · 2021 · $343,606

## Abstract

The mitochondrial inner membrane is the site of essential cellular functions such as oxidative
phosphorylation, phospholipid metabolism, and the regulation of apoptosis. These activities are
performed by a composite mitochondrial proteome that requires constant resculpting to respond
to both the changing metabolic demands of the cell and the emergence of damage driven by
reactive oxygen species. This resculpting is performed by two mitochondrial AAA+ proteases,
which harness the energy of ATP to recognize, unfold and degrade protein substrates both from
within and surrounding the inner membrane. In humans, dysfunction of these proteases has been
linked to the development of severe neurodegenerative disorders such as spinocerebellar ataxia.
AAA+ proteases assemble as hexamers to form an internal proteolytic chamber into which
substrates are forcibly translocated by a ring of ATPases. The study of the mitochondrial AAA+
proteases has been long hampered by their combination of multiple soluble catalytic domains with
insoluble transmembrane domains for anchoring into the inner membrane. We utilize a protein-
engineering approach to assemble previously membrane-constrained hexameric proteases in a
soluble, active form. Our goal is to use these rebuilt proteases to perform a rigorous analysis of
the mechanisms driving energy-dependent proteolysis at the mitochondrial inner membrane. The
first aim of the proposal is to define how substrates are selected for degradation among the myriad
mitochondrial proteins. Degradation signal sequences will be identified from physiological
substrates to ask whether these signals are conserved across diverse mitochondrial proteins to
enable recognition by common proteases. The second aim is to examine the recognition complex
formed between these proteases and specific substrates. A series of complementary biochemical
approaches will map the protease substrate binding sites and identify the complementary contacts
used to promote selection and degradation. Finally, we will examine how the architecture of the
proteolytic sites within the degradation chamber achieves specificity of peptide-bond cleavage
specificity, resulting in site-specific cleavage of a class of substrates, including the regulator of
mitochondrial fission. Together, these experiments will provide a rigorous mechanistic analysis of
the mitochondrial AAA+ proteases and provide foundational knowledge to aid the development
of small molecule modulators as future therapeutics.

## Key facts

- **NIH application ID:** 10296122
- **Project number:** 2R01GM115898-06A1
- **Recipient organization:** STATE UNIVERSITY NEW YORK STONY BROOK
- **Principal Investigator:** Steven Glynn
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $343,606
- **Award type:** 2
- **Project period:** 2015-07-15 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10296122

## Citation

> US National Institutes of Health, RePORTER application 10296122, Using rebuilt AAA+ enzymes to uncover the mechanisms of proteolysis at the mitochondrial inner membrane (2R01GM115898-06A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10296122. Licensed CC0.

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