Biology subjects

Clancy, A.

Publications and source records attributed to Clancy, A..

3 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↗

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↗

bioRxiv2020

The deubiquitylase USP9X controls ribosomal stalling

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases respectively, through ubiquitylation of the eS10 and uS10 sub-units of the ribosome. It is common for the stability of RING E3 ligases to be regulated by an interacting deubiquitylase (DUB), which often opposes auto-ubiquitylation of the E3. Here, we show that the DUB USP9X directly interacts with ZNF598 and regulates its abundance through the control of protein stability in human cells. We have developed a highly specific small molecule inhibitor of USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome associated protein stability and reveals loss of ZNF598 and Makorin 2. In the absence of USP9X or following chemical inhibition of its catalytic activity, steady state levels of Makorins and ZNF598 are diminished and the ribosomal quality control pathway is impaired.

cell biology↗