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.
INDRA was used to automatically assemble known mechanisms
related to USP35 from literature and knowledge bases.
The first section shows only DUB activity and the second shows all other results.
Collectively, our data indicated that miR let-7a-regulated USP35 can inhibit NF-kappaB activation by deubiquitination and stabilization of ABIN-2 protein and eventually inhibit cell proliferation.
Together, these data illustrated that USP35 could up-regulate the expression of ABIN-2 by promoting ABIN-2 deubiquitination and decreasing its proteasomal degradation.
Recently, we have found that USP35 acts as a deubiquitinating enzyme (DUB) for Aurora B and affects its stability during cell division, thus being involved in the regulation of mitosis.
Mechanistically, USP35 enhanced ERalpha stability by interacting and deubiquitinating ERalpha, and transcriptional activity of ERalpha by interacting with ERalpha in DNA regions containing estrogen response element.
Liu and colleagues have reported that USP35 inhibits cell proliferation via suppression of NF-κB activation by deubiquitination and stabilization of ABIN-2 (Liu C et al (2015) Oncotarget 6, 27891–27906).
We then determined the roles of USP35 and found that overexpression of USP35 could inhibit cancer cell proliferation in vitro and tumorigenesis in vivo.
MTT results showed that overexpression of USP35 inhibited cell proliferation significantly compared with their respective controls in both H1299 and LNCaP cells (Figure xref ).
MTT results showed that overexpression of USP35 inhibited cell proliferation significantly compared with their respective controls in both H1299 and LNCaP cells.
The results showed that overexpression of USP35 significantly extended the half-life of ABIN-2, while silenced expression of USP35 shortened the half-life of ABIN-2, indicating that USP35 promotes ABIN-2 protein stabilization.
Together, these data illustrated that USP35 could up-regulate the expression of ABIN-2 by promoting ABIN-2 deubiquitination and decreasing its proteasomal degradation.
Functionally, both ABIN-2 and USP35 could inhibit TNFα-induced NF-κB activation and overexpression of ABIN-2 alleviated USP35-loss induced activation of NF-κB. Collectively, our data indicated that miR let-7a-regulated USP35 can inhibit NF-κB activation by deubiquitination and stabilization of ABIN-2 protein and eventually inhibit cell proliferation.
XREF_BIBR In addition, miR-let-7a was shown to regulate USP35 expression, which then inhibited NF-kappaB activation by deubiquitination and stabilization of TNIP2 protein.
Collectively, our data indicated that miR let-7a-regulated USP35 can inhibit NF-kappaB activation by deubiquitination and stabilization of ABIN-2 protein and eventually inhibit cell proliferation.
Although, USP35 is not a predicted target of miR let-7a by the web based bioinformatic analysis, the fact from our experiment is that the expression of USP35 can be up-regulated by miR let-7a.
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].
Western blot analysis also showed that the depletion of USP35 lowered the levels of both phospho-histone H3 and Aurora B protein in nocodazole treated cells.
Since it is known that the ubiquitination of Aurora B regulates its stability or localization XREF_BIBR, XREF_BIBR, we then tested whether USP35 could perturb the protein levels or localization of Aurora B.
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]
USP35 inhibits CDH1 mediated degradation of Aurora B. Having noted the effect of USP35 depletion on the levels of Aurora B protein, we expected that USP35 could deubiquitinate Aurora B, and counteract the ubiquitination activity of APC CDH1 E3 ligase.