Biology subjects

Rusilowicz-Jones, E. V.

Publications and source records attributed to Rusilowicz-Jones, E. V..

4 recordsLinked to original sources

bioRxiv2026

Arrested PPTC7 import couples mitochondrial depolarisation to NIX and BNIP3 degradation

Mitophagy is initiated by two distinct pathways that are both coupled to mitochondrial protein import. Import stress stabilises PINK1 at the outer mitochondrial membrane, activating the Parkin and ubiquitin-dependent pathway. Receptor-mediated pathways are governed by the expression of outer mitochondrial membrane proteins NIX and BNIP3. Mitophagy is limited by their degradation, via SCFFBXL4. This requires the PP2C-family phosphatase PPTC7 as an obligate substrate recognition factor, captured at the outer membrane en route to the matrix. We show that mitochondrial depolarisation exerts opposing effects on these two pathways. In RPE1 cells, depolarisation drives rapid, ubiquitin and proteasome-dependent loss of NIX and BNIP3 that requires ongoing protein synthesis of PPTC7. Using a doxycycline-mediated pulse-chase assay, we demonstrate directly that depolarisation inhibits import of newly synthesised PPTC7, and that this arrested PPTC7 is itself degraded in an FBXL4-dependent manner alongside its substrates. Thus, import arrest both stabilises PINK1 and eliminates NIX and BNIP3, coordinating a switch between mitophagy pathways, while concurrent degradation of PPTC7 couples mitophagy receptor turnover to PPTC7 synthesis rates.

cell biology↗

bioRxiv2024

A long-lived pool of PINK1 imparts a molecular memory of depolarisation-induced activity.

The Parkinsons disease linked kinase, PINK1, is a short lived protein that undergoes cleavage upon mitochondrial import leading to its release to the cytosol and proteasomal degradation. Under mitochondria depolarising conditions, it accumulates on mitochondria where it becomes activated, phosphorylating both ubiquitin and the ubiquitin E3 ligase Parkin, at Ser65. Here we have used a ubiquitylation inhibitor TAK-243 to accumulate cleaved PINK1 (cPINK1) in a cell line that lacks Parkin. We show that cPINK1 phosphorylates free ubiquitin and can be released to the cytosol in an active form. We show that in RPE1 cells under mitochondria depolarising conditions (i) the majority of PINK1 cleavage proceeds unimpeded and (ii) accrued PINK1 cannot be accounted for by protein stabilisation alone. Accordingly, we suggest that translation of PINK1 mRNA must be mobilised under mitochondrial depolarisation. We have further discovered a pre-conditioning phenomenon, whereby an initial depolarising treatment leaves a residual pool of active PINK1, which remains competent for seeding the activation of nascent cPINK1, despite a 16 hour recuperation period.

cell biology↗

bioRxiv2023

ISGylation-independent protection of cell growth by USP18 following interferon stimulation

Type 1 interferon stimulation highly up-regulates all elements of a ubiquitin-like conjugation system that leads to ISGylation of target proteins. An ISG15-specific member of the deubiquitylase family, USP18, is up-regulated in a co-ordinated manner. USP18 can also provide a negative feedback by inhibiting JAK-STAT signaling through protein interactions independently of DUB activity. Here, we provide an acute example of this phenomenon, whereby the early expression of USP18, post-interferon treatment of HCT116 colon cancer cells is sufficient to fully suppress the expression of the ISG15 E1 enzyme, UBA7. Stimulation of lung adenocarcinoma A549 cells with interferon reduces their growth rate but they remain viable. In contrast, A549 USP18 knock-out cells show similar growth characteristics under basal conditions, but upon interferon stimulation a profound inhibition of cell growth is observed. We show that this contingency on USP18 is independent of ISGylation, suggesting non catalytic functions are required for viability. We also demonstrate that global deISGylation kinetics are very slow compared with deubiquitylation. This is not influenced by USP18 expression, suggesting that enhanced ISGylation in USP18 KO cells reflects increased conjugating activity.

cell biology↗

bioRxiv2021

Benchmarking a highly selective USP30 inhibitor for enhancement of mitophagy and pexophagy.

The deubiquitylase USP30 is an actionable target considered for treatment of conditions associated with defects in the PINK1/Parkin pathway leading to mitophagy. These include Parkinsons disease and pulmonary fibrosis. We provide a detailed cell biological characterisation of a benzenesulphonamide molecule, compound 39, that has previously been reported to inhibit USP30 in an in vitro enzymatic assay. The current compound offers increased selectivity over previously described inhibitors. It enhances mitophagy and generates a signature response for USP30 inhibition following mitochondrial depolarisation. This includes enhancement of TOM20 and SYNJ2BP ubiquitylation and phosphoubiquitin accumulation, alongside increased mitophagy. In dopaminergic neurons, generated from Parkinsons disease patients carrying loss of function Parkin mutations, compound 39 could significantly restore mitophagy to a level approaching control values. USP30 is located on both mitochondria and peroxisomes and has also been linked to the PINK1 independent pexophagy pathway. Using a fluorescence reporter of pexophagy expressed in U20S cells, we observe increased pexophagy upon application of compound 39 that recapitulates the previously described effect for USP30 depletion. This provides the first pharmacological intervention with a synthetic molecule to enhance peroxisome turnover.

cell biology↗