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 USP50 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 USP50 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
However, the Flag-USP50 (C53S) mutant, did not affect polyubiquitination of the ASC protein, indicating that the Cys residue at position 53 is critical for the catalytic activity of USP50 deubiquitination of the ASC protein.
Ubiquitination of Flag-ASC proteins was significantly decreased in the presence of Flag-USP50 protein, indicating that USP50 directly deubiquitinates the polyubiquitination of the ASC protein.
In conclusion, we here demonstrate a novel regulatory mechanism regarding the ASC protein which is deubiquitinated by USP50 and provide experimental evidence emphasizing the importance of a regulatory mechanism mediated by ubiquitination and deubiquitination in inflammasome activation.
Lee et al.66 showed that the deubiquitinating enzyme, USP50, binds to and deubiquitinates lysine 63‐linked polyubiquitin chains of ASC and that this is required for NLRP3 inflammasome activation, since USP50‐deficient cells show impaired inflammasome activation.
Lee et al.66 showed that the deubiquitinating enzyme, USP50, binds to and deubiquitinates lysine 63‐linked polyubiquitin chains of ASC and that this is required for NLRP3 inflammasome activation, since USP50‐deficient cells show impaired inflammasome activation.
We propose that USP50 may act through a HSP90 dependent mechanism to counteract CDC25B mitotic inducing activity and prevent Wee1 degradation, thereby repressing entry into mitosis following activation of the DNA damage checkpoint.
In response to DNA damage, USP50 accumulates in the nucleus and may act through an HSP90 dependent mechanism to counteract CDC25B mitotic inducing activity and prevent Wee1 degradation, thereby repressing entry into mitosis following activation of the DNA damage checkpoint [XREF_BIBR].
USP50 (ubiquitin specific protease 50), a deubiqitinating enzyme (DUB), prevents Wee1 degradation, thereby repressing entry into mitosis following activation of the G2/M DNA damage checkpoint.
We also demonstrate USP50 depletion causes a loss in accumulation of the HSP90 client Wee1, which is an essential component of the G2/M cell cycle arrest.
Taken together with our results that USP50 depletion decreases the formation of ASC specks as well as the secretion of IL-lbeta, our findings strongly suggest the possibility that removal of these polyubiquitin chains from ASC protein by USP50 is a crucial step for inflammasome activation.
In contrast, USP50 depletion significantly decreased IL-1beta secretion, suggesting the possibility that USP50 is a positive regulator in inflammasome activation.
Curcumin alleviated LPS-mediated sepsis by suppressing the activities of mitochondrial STAT3 and NF-κB. Conclusion: Our findings reveal that mitochondrial STAT3 could trigger FAO by inducing CPT1a stabilization mediated by USP50 in macrophages, at least partially.