USP30 sets a trigger threshold for PINK1–PARKIN amplification of mitochondrial ubiquitylation

Rusilowicz‐Jones, Emma V; Jardine, Jane; Kallinos, Andreas; Pinto-Fernández, Adán; Guenther, Franziska; Giurrandino, Mariacarmela; Barone, Francesco G.; McCarron, Katy R; Burke, Christopher; Murad, Alejandro D.; Amador, Martínez; Marcassa, Elena; Gersch, Malte; Buckmelter, Alexandre J.; Kayser-Bricker, Katherine J.; Lamoliatte, Frederic; Gajbhiye, Akshada; Davis, Simon; Scott, Hannah C.; Murphy, Emma J.; England, Katherine S.; Mortiboys, Heather; Komander, David; Trost, Matthias; Kessler, Benedikt M.; Ioannidis, Stephanos; Ahlijanian, Michael K.; Urbé, Sylvie; Clague, Michael J.

doi:10.26508/lsa.202000768

Cited by 88 publications

(105 citation statements)

References 68 publications

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“…Several candidates have been considered in this regard, with USP30 currently the most well-studied. USP30 is a mitochondria-anchored DUB that preferentially hydrolyses Lys6-linked ubiquitin chains on mitochondrial substrates shared with Parkin to set a threshold for mitophagy [ 35 , 37 – 39 , 200 ]. In a key study, Bingol et al showed that USP30 overexpression limited PINK1/Parkin-mediated mitophagy in cells.…”

Section: Targeting Ubiquitin Signalling To Treat Neurodegenerative DImentioning

confidence: 99%

“…Kluge et al reported the phenylalanine derivative MF-094 as a selective inhibitor of USP30, which accelerated mitophagy in C2C12 myotubes [ 202 ]. Recently, Rusilowicz-Jones et al reported a N-cyano pyrrolidine derivative (FT3967385) inhibited USP30 with an IC 50 of 1.5 nM and, whilst it showed some off-target inhibition of USP6, enhanced mitophagy in cultured neuroblastoma cells [ 200 ] (Fig. 4B ).…”

Section: Targeting Ubiquitin Signalling To Treat Neurodegenerative DImentioning

confidence: 99%

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Ubiquitin signalling in neurodegeneration: mechanisms and therapeutic opportunities

et al. 2021

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Neurodegenerative diseases are characterised by progressive damage to the nervous system including the selective loss of vulnerable populations of neurons leading to motor symptoms and cognitive decline. Despite millions of people being affected worldwide, there are still no drugs that block the neurodegenerative process to stop or slow disease progression. Neuronal death in these diseases is often linked to the misfolded proteins that aggregate within the brain (proteinopathies) as a result of disease-related gene mutations or abnormal protein homoeostasis. There are two major degradation pathways to rid a cell of unwanted or misfolded proteins to prevent their accumulation and to maintain the health of a cell: the ubiquitin–proteasome system and the autophagy–lysosomal pathway. Both of these degradative pathways depend on the modification of targets with ubiquitin. Aging is the primary risk factor of most neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. With aging there is a general reduction in proteasomal degradation and autophagy, and a consequent increase of potentially neurotoxic protein aggregates of β-amyloid, tau, α-synuclein, SOD1 and TDP-43. An often over-looked yet major component of these aggregates is ubiquitin, implicating these protein aggregates as either an adaptive response to toxic misfolded proteins or as evidence of dysregulated ubiquitin-mediated degradation driving toxic aggregation. In addition, non-degradative ubiquitin signalling is critical for homoeostatic mechanisms fundamental for neuronal function and survival, including mitochondrial homoeostasis, receptor trafficking and DNA damage responses, whilst also playing a role in inflammatory processes. This review will discuss the current understanding of the role of ubiquitin-dependent processes in the progressive loss of neurons and the emergence of ubiquitin signalling as a target for the development of much needed new drugs to treat neurodegenerative disease.

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Section: Targeting Ubiquitin Signalling To Treat Neurodegenerative DImentioning

confidence: 99%

Section: Targeting Ubiquitin Signalling To Treat Neurodegenerative DImentioning

confidence: 99%

Ubiquitin signalling in neurodegeneration: mechanisms and therapeutic opportunities

et al. 2021

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“…Therefore, we determined changes in mitophagy by measuring the mitoKeima signal in cells treated with USP30Inh-1 at the time point of 3 days. Prolonged incubation might be required in order to see changes in mitophagy after inhibition of USP30 which could be due to its suggested role in controlling the threshold of mitophagy (Rusilowicz-Jones et al, 2020, Marcassa et al, 2018. Next, SHSY5Y mitoKeima cells were treated with 0.1, 1 and 10 μM USP30Inh-1 for 3 days.…”

Section: Pharmacological Inhibition Of Usp30 Enhances Mitokeima Signamentioning

confidence: 99%

Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches

Ketteler

Tsefou

Walker

et al. 2021

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Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterization of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 in the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared to wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.

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“…The assay was carried out according to [ 48 ]. MITOL-dependent ubiquitylation was standardized in a ubiquitylation–deubiquitylation buffer (20 mM Tris-Cl pH 8.0, 0.01% Triton X-100, 1 mM L-glutathione, 0.03% BSA, 5 mM MgCl2, and 2 mM ATP).…”

Section: Methodsmentioning

confidence: 99%

MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria

et al. 2021

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Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.

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USP30 sets a trigger threshold for PINK1–PARKIN amplification of mitochondrial ubiquitylation

Cited by 88 publications

References 68 publications

Ubiquitin signalling in neurodegeneration: mechanisms and therapeutic opportunities

Ubiquitin signalling in neurodegeneration: mechanisms and therapeutic opportunities

Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches

MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria

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