The Dependency Map (DepMap) is a genome-wide pooled
CRISPR-Cas9 knockout proliferation screen conducted in more than 700 cancer cell lines spanning many
different tumor lineages. Each cell line in the DepMap contains a unique barcode, and each gene
knockout is assigned a “dependency score” on a per cell-line basis which quantifies the rate of
CRISPR-Cas9 guide drop. It has been found that proteins with similar DepMap scores across cell
lines, a phenomenon known as co-dependent genes, have closely related biological functions. This can
include activity in the same or parallel pathways or membership in the same protein complex or the
same pathway.
We identified the strongest seven co-dependent genes (“Symbol”) for DUBs and ran GO enrichment
analysis. We used Biogrid, IntAct, and Pathway Commons PPIDs, and the NURSA protein-protein
interaction databases (PPIDs) to determine whether co-dependent genes interact with one another. The
“Evidence” column contains the PPIDs in which the interaction appears as well as whether there is
support for the association by an INDRA statement. As another approach to identify potential
interactors, we looked at proteomics data from the Broad Institute's Cancer Cell Line Encyclopedia (CCLE) for
proteins whose expression across ~375 cell lines strongly correlated with the abundance of each DUB;
it has previously been observed that proteins in the same complex are frequently significantly
co-expressed. The correlations and associated p-values in the CCLE proteomics dataset are provided.
And, we determined whether co-dependent genes yield similar transcriptomic signatures
in the Broad Institute's Connectivity
Map (CMap). A CMap score greater than 90 is considered significantly similar.
Using the biological processes and other Gene Ontology terms from well characterized DUBs as a
positive control, several gene set enrichment analyses were considered. Threshold-less methods
like GSEA had relatively poor results.
Over-representation analysis with a threshold of of the top 7 highest absolute value Dependency Map
correlations yielded the best results and is reported below.
GO Identifier
GO Name
GO Type
p-value
p-value (adj.)
q-value
Transcriptomics
The following table shows the significantly differentially expressed genes after knocking
out USP30 using CRISPR-Cas9.
There were too few differentially expressed genes to run a meaningful GSEA.
Literature Mining
INDRA was used to automatically assemble known mechanisms
related to USP30 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
USP30, a mitochondrial deubiquitinase, promotes mitochondrial fusion by mediating the deubiquitination of ubiquitylated forms of mitofusins, such as Mfn1 and Mfn2.
USP30, a mitochondrial deubiquitinase, promotes mitochondrial fusion by mediating the deubiquitination of ubiquitylated forms of mitofusins, such as Mfn1 and Mfn2.
Mechanistically, GNPAT recruited the enzyme USP30, which deubiquitylated and stabilized dynamin related protein 1 (DRP1), thereby facilitating regulation of mitochondrial morphology, lipid metabolism, and hepatocarcinogenesis.
Similar findings were reported for another DUB: USP30. USP30 overexpression impaired PARKIN-mediated mitophagy by deubiquitinating mitochondrial substrates that include TOM20, TOM70, and the VDACs
As expected, expression of WT USP30, but not a catalytically inactive mutant (C77A), reversed TOMM20 ubiquitylation, but did not affect MFN2 or CISD1 ubiquitylation (XREF_FIG, XREF_SUPPLEMENTARY).
Parkin mediated effects are further regulated by de-ubiquitinating enzymes, such as USP30 and USP35, which reportedly de-ubiquitinate Parkin substrates to antagonize mitophagy [24 *] or Parkin depende[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Parkin mediated effects are further regulated by de-ubiquitinating enzymes, such as USP30 and USP35, which reportedly de-ubiquitinate Parkin substrates to antagonize mitophagy [XREF_BIBR] or Parkin dependent cell death [XREF_BIBR].
However, whether USP30 also comprises specificity at the level of primary ubiquitylation sites in MOM substrates, and the extent to which USP30 activity suppresses the PARKIN feed-forward activation mechanism via pS65-Ub has not been examined in more physiological systems such as neurons.
In contrast, a recent report demonstrated that in Drosophila the age dependent increase in mitophagy in both muscle and dopaminergic neurons is dependent on PINK1 and Parkin, and the knockdown of USP15 and USP30 rescues mitophagy in Parkin deficient organisms.
On one hand, overexpression of USP30 can block Parkin dependent accumulation of Ub chains on MOM proteins in response to depolarization, suggesting that USP30 directly antagonizes Parkin activity.
The best evidence to date comes from analyzing USP30 which appears to antagonize Parkin function as evidenced by the fact that genetic inhibition of USP30 rescues Parkin deficient flies.
Strikingly USP30 knockdown invivo could rescue the PINK1 or Parkin -/- phenotypes in Drosophila, indicating that the inhibition of USP30 could be therapeutically advantageous in patients with equivalent null mutations in these genes [XREF_BIBR].
Emerging promising molecules include selective inhibitor of the mitochondrial deubiquitinase, USP30 that negatively regulates PRKN-mediated mitophagy [132,200].
Early studies on USP30 focused on reversal of Parkin dependent MOM ubiquitylation in HeLa cells overexpressing Parkin, as well as in Drosophila, where reduction in USP30 function enhanced the activity of Parkin mutants.
Although SILAC provides a rigorous way for performing quantification, other studies used label-free quantification to examine targeted mitochondrial outer membrane ubiquitylation by PARKIN in the pres[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Although SILAC provides a rigorous way for performing quantification, other studies used label-free quantification to examine targeted mitochondrial outer membrane ubiquitylation by PARKIN in the presence or absence of the mitochondrial deubiquitylating enzyme USP30, leading to the identification of a dozen mitochondrial PARKIN targets that are regulated by USP30 (see below).
Knockdown of the Drosophila homologs of USP15 (CG8334, hereafter called dUSP15) and USP30 (CG3016, hereafter dUSP30) largely rescues the mitochondrial defects of parkin deficient fly muscle in vivo.
Knockdown of the mitochondrial deubiquitinase, USP30, rescues mitophagy defects and disease in flies with pathogenic mutations in Parkin, suggesting a potential role for the inhibition of DUBs that target selective autophagy E3 ligases in the treatment of Parkinson 's and other diseases.
Knockdown of USP30 rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1 deficient flies.
Parkin mediated effects are further regulated by de-ubiquitinating enzymes, such as USP30 and USP35, which reportedly de-ubiquitinate Parkin substrates to antagonize mitophagy [24 *] or Parkin depende[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
In contrast, direct siRNA mediated depletion of MIRO2 to comparable residual levels marginally decreased PARP cleavage, whilst a combined knockdown of USP30 and MIRO2 promoted cell death in a similar fashion to USP30 siRNA on its own (XREF_SUPPLEMENTARY).
Cell death promoted by USP30 depletion upon treatment with ABT-737 was partially suppressed by BAX knockdown but required concomitant depletion of both BAK and BAX for full suppression (Fig XREF_FIG D and E).
Our data showing that USP30 suppresses a PINK1 dependent component of basal mitophagy indicate a link between USP30 function and phospho-ubiquitin, the key substrate of PINK1 in mitophagy.
One model consistent with this observation envisages that by suppressing basal ubiquitylation at mitochondria, USP30 may effectively limit PINK1 ubiquitin-substrate availability and the generation of pUb ' Parkin-receptor sites ', thus primarily influencing the initiation phase of mitophagy 86.
Strikingly USP30 knockdown invivo could rescue the PINK1 or Parkin -/- phenotypes in Drosophila, indicating that the inhibition of USP30 could be therapeutically advantageous in patients with equivalent null mutations in these genes [XREF_BIBR].
Here we report that USP30, a deubiquitinase localized to mitochondria, antagonizes mitophagy driven by the ubiquitin ligase parkin (also known as PARK2) and protein kinase PINK1, which are encoded by two genes associated with Parkinson 's disease.
Knockdown of USP30 rescues the defective mitophagy caused by pathogenic mutations in parkin and improves mitochondrial integrity in parkin- or PINK1 deficient flies.
Although SILAC provides a rigorous way for performing quantification, other studies used label-free quantification to examine targeted mitochondrial outer membrane ubiquitylation by PARKIN in the presence or absence of the mitochondrial deubiquitylating enzyme USP30, leading to the identification of a dozen mitochondrial PARKIN targets that are regulated by USP30 (see below).
Although SILAC provides a rigorous way for performing quantification, other studies used label-free quantification to examine targeted mitochondrial outer membrane ubiquitylation by PARKIN in the pres[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Whilst the phosphatase that dephosphorylates p-Ub remains unknown, two DUBs have been identified that deubiquitylate Parkin directed substrates, USP30 and USP15, and USP8 has also been reported to reverse Parkin autoubiquitylation.
One potential explanation for no effect on the feed-forward process with overt depolarization is that activated Parkin rapidly inactivates USP30 or otherwise outpaces Ub removal by USP30, as previously proposed.
Thus, USP30 may enable a dynamic ubiquitin economy that is required for multiple core functions at these organelles, whilst preventing inadvertent engagement of the autophagy machinery.
The ubiquitin chains targeted by USP30 are similar to the ones Parkin adds to its substrates, suggesting that these two enzymes act as antagonists on shared substrates.
Overexpression of USP30 prevents pexophagy during amino acid starvation, by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2 [266, 267].
Overexpression of USP30 prevents pexophagy during amino acid starvation, by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2 XREF_BIBR, XREF_BIBR.
Overexpression of USP30 prevents pexophagy during amino acid starvation, by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2 XREF_BIBR, XREF_BIBR.
Overexpression of USP30 prevents pexophagy during amino acid starvation, by counteracting the action of the peroxisomal E3 ubiquitin ligase PEX2 [266, 267].
The inhibition of USP30 by S3 leads to an increase of non degradative ubiquitination of Mfn1/2, which enhances Mfn1 and Mfn2 activity and promotes mitochondrial fusion.
In this regard, we further observed that co-depletion of STK38 and USP30 was sufficient to fully restore total mitochondrial mass and mitochondrial ROS levels to normal values as observed in control cells.
Even more importantly, depletion of USP30, a major opponent of PINK1 and Parkin mediated mitophagy [XREF_BIBR, XREF_BIBR], partially restored soft agar growth, and fully restored total mitochondrial mass and ROS levels of STK38 depleted Ras transformed cells.
S3 induced inhibition of USP30, a mitochondrial localized deubiquitinase, increased the ubiqutinylation of MFN1 and MFN2 without affecting their protein levels.
For example, based on a phenotypic screening, it was shown that the inhibition of USP30 by the compound 15-oxospiramilactone enhances the activity of USP30 's targets Mfn1 and Mfn2 -- two GTPases anchored at the OMM and essential for tethering adjacent mitochondria -- and promotes mitochondrial fusion, thus contributing to the restoration of the mitochondria network [XREF_BIBR].
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.
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 [XREF_BIBR].
One model consistent with this observation envisages that by suppressing basal ubiquitylation at mitochondria, USP30 may effectively limit PINK1 ubiquitin-substrate availability and the generation of pUb ' Parkin-receptor sites ', thus primarily influencing the initiation phase of mitophagy 86.
RNAi mediated knockdown of USP30 specifically in dopaminergic neurons via the dopamine decarboxylase driver completely rescued the paraquat induced behavioral deficit and prevented dopamine depletion [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]
Structure of a USP20 inhibitor from GSK.Figure 29 Structure of a USP30 inhibitor.Structures of UCHL1 inhibitors.VAE(OMe)-FMK Structure of a weak tripeptide FMK UCHL1 inhibitor.Figure 33 Structures of UCHL3 inhibitors.Structures of TRABID and RPN11 inhibitors.
As expected, Cys and His mutations abrogate USP30 activity, while Ser mutation to Ala, but also to Asn or Asp, that are usually found in USP DUBs XREF_BIBR, reduce activity in both mono- and diubiquitin based substrate cleavage assays (XREF_FIG, XREF_SUPPLEMENTARY).
In this regard, we speculated that USP30 depletion might compensate for STK38 deficiency, hence restoring anchorage independent growth of STK38 depleted Ras transformed cells.
S3 induced inhibition of USP30, a mitochondrial localized deubiquitinase, increased the ubiqutinylation of MFN1 and MFN2 without affecting their protein levels.
In contrast, a systematic analysis of DUB knockdown phenotypes in Drosophila highlighted a protective role for USP30 during development : even incomplete suppression of USP30 expression caused developmental lethality or early death in adulthood [XREF_BIBR].
We further reconstitute USP30 regulatory mechanisms in vitro, and show how even incomplete PINK1 kinase mediated ubiquitin chain phosphorylation impacts USP30 activity.
Enhancement of MFN1 activity by S3 mediated inhibition of USP30 (mitochondria localized deubiquitinase) restored mitochondrial morphology and ATP levels in fusion deficient mouse embryo fibroblasts (Yue etal., 2014).