# Regulation of PINK1 and PARKIN-dependent mitophagy

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2021 · $439,412

## Abstract

SUMMARY. Numerous studies, including critical work from our lab, has revealed the fundamental mechanisms
by which proteins encoded by two Parkinson’s Disease (PD) genes – the PINK1 protein kinase and PARKIN
ubiquitin (Ub) ligase – promote the ubiquitylation and autophagic capture of damaged mitochondria to promote
their clearance by mitophagy. Recently, we have merged a quantitative proteomics platform with stem cell-
derived, induced neurons (iNeurons) harboring pathway mutations to elucidate PARKIN and PINK1 ubiquitylation
targets under endogenous conditions, and have determined the role of the mitochondrial deubiquitylase USP30
and the p97 segregase in PARKIN and mitophagic flux regulation. Yet, our understanding of the extent to which
other proteins mutated in PD collaborate with the PARKIN-PINK1 system to contribute to disease etiology
remains limited, as is our understanding of how the PINK1 activation threshold on the mitochondrial translocon
is mechanistically controlled. Here, we propose a series of experiments that address both of these knowledge
gaps. First, among the most compelling genes to emerge from our recent mitophagic flux CRISPR screen is
FBXO7, a gene mutated in PD (PARK15) and a member of the F-Box family of proteins that forms an SCF Ub
ligase. FBXO7’s critical functions and targets, as well as how its mutation predisposes to PD, are unknown.
Through interaction proteomics, we find that FBXO7 associates with multiple regulatory components of the
proteasome, and propose that FBXO7 may play a central role by integrating mitophagy and proteasomal control
mechanisms to support organelle homeostasis. In Aim 1, we will use our iNeuron system to examine FBXO7’s
role in mitophagic flux using an array of quantitative assays that examine sequential steps in the pathway, and
we will genetically and functionally dissect ubiquitylation targets and regulatory mechanisms as an initial step
toward understanding how patient mutations in FBXO7 may contribute to PD. Second, our preliminary data, and
work in the field, indicate that both PINK1 and USP30 are physically associated with the mitochondrial translocon,
placing the translocon at the nexus of PARKIN regulation. Our data show that USP30 has a role in controlling
both the threshold for PARKIN activation by removing Ub from the translocon and also may have a previously
unappreciated role in import quality control at the translocon itself. In Aim 2, we will systematically examine
translocon components and ubiquitylation for their roles in setting the threshold for PARKIN activation via Ub
phosphorylation. In parallel, we will elucidate how USP30 functions in this newly recognized Import Quality
Control (IQC) pathway for removal of Ub chains from translocon import substrates. Finally, our work has led to
the first visualization of PINK1 in association with the translocon using single-particle electron microscopy, and
we seek to further develop a biochemical and structural understanding of ...

## Key facts

- **NIH application ID:** 10212467
- **Project number:** 5R01NS083524-17
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** JEFFREY W HARPER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $439,412
- **Award type:** 5
- **Project period:** 2013-09-25 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10212467

## Citation

> US National Institutes of Health, RePORTER application 10212467, Regulation of PINK1 and PARKIN-dependent mitophagy (5R01NS083524-17). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10212467. Licensed CC0.

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