Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the ...interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.
The SYK non-receptor tyrosine kinase is a key effector of immune receptors signaling in hematopoietic cells. Here, we identified and characterized a novel interaction between SYK and the ...ubiquitin-specific protease 25 (USP25). We report that the second SH2 domain of SYK physically interacts with a tyrosine-rich, C-terminal region of USP25 independently of tyrosine phosphorylation. Moreover, we showed that SYK specifically phosphorylates USP25 and alters its cellular levels. This study thus uncovers a new SYK substrate and reveals a novel SYK function, namely the regulation of USP25 cellular levels.
The homeodomain protein TGIF (TG-interacting factor) restricts TGF-β/Smad cytostatic signaling by interfering with the nucleocytoplasmic transit of the tumor suppressor cPML. Here, we identify PHRF1 ...as a ubiquitin ligase that enforces TGIF decay by driving its ubiquitination at lysine 130. In so doing, PHRF1 ensures redistribution of cPML into the cytoplasm, where it associates with SARA and coordinates activation of Smad2 by the TGF-β receptor. The PHRF1 gene resides within the tumor suppressor locus 11p15.5, which displays frequent loss in a wide variety of malignancies, including breast cancer. Remarkably, we found that the PHRF1 gene is deleted or silenced in a high proportion of human breast cancer samples and cancer cell lines. Reconstitution of PHRF1 into deficient cells impeded their propensity to form tumors in vivo, most likely because of the reemergence of TGF-β responsiveness. These findings unveil a paradigm behind inactivation of the cPML tumor suppressor network in human malignancies.
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•PHRF1 functions as a ubiquitin ligase to target TGIF for degradation•PHRF1 empowers TGF-β cytostatic signaling•PHRF1 promotes TGF-β/Smad signaling by ensuring cytoplasmic relocalization of cPML•PHRF1 bears the hallmarks of a tumor suppressor gene in breast cancer
Recurrent inactivation of the TGF-β cytostatic program is a critical determinant of tumor pathogenesis and progression. Atfi and colleagues now demonstrate that the ubiquitin ligase PHRF1 functions as a mediator of TGF-β signaling by targeting TGIF for degradation. In so doing, PHRF1 empowers the tumor suppressor cPML to orchestrate initiation and activation of Smad signaling. This PHRF1 function is prone to disruption in cancer, as exemplified by the frequent deletion or silencing of the PHRF1 gene in human breast cancers.
Deregulation of the ubiquitin-proteasome system has been implicated in the pathogenesis of many human diseases, including cancer, neurodegenerative disorders and viral diseases. The recent approval ...of the proteasome inhibitor bortezomib (Velcade
®) for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. A promising alternative to targeting the proteasome itself would be to interact at the level of the upstream, ubiquitin conjugation/deconjugation system to generate more specific, less toxic anticancer agents. Ubiquitin specific proteases (USP) are de-ubiquitinating enzymes which remove ubiquitin from specific protein substrates and allow protein salvage from proteasome degradation, regulation of protein localization or activation. Due to their protease activity and their involvement in several pathologies, USPs are emerging as potential target sites for pharmacological interference in the ubiquitin regulatory machinery. We will review here this class of enzymes from target validation to small molecule drug discovery.
Many types of human cancers having hyperactivated Wnt signaling display no causative alterations in known effectors of this pathway. Here, we report a function of TGIF in Wnt signaling. TGIF ...associates with and diverts Axin1 and Axin2 from the β-catenin destruction complex, therefore allowing β-catenin accrual. Intriguingly, activation of Wnt signaling induces the expression of TGIF, which unveils a feed-forward loop that ensures effective integration of Wnt signaling. In triple-negative breast cancers (TNBC), elevated levels of TGIF correlate with high Wnt signaling and poor survival of patients. Moreover, genetic experiments revealed that Tgif1 ablation impeded mammary tumor development in MMTV-Wnt1 mice, further underscoring a requirement of TGIF for oncogenic Wnt signaling.
•TGIF functions as a mediator of Wnt/β-catenin signaling•The association of TGIF with Axin1 and Axin2 promotes β-catenin stability•TGIF1 is a Wnt target gene•TGIF is essential for Wnt-driven mammary tumorigenesis
Zhang et al. show that TGIF interacts with Axin1 and Axin2, thus preventing them from translocating to cytoplasm to form the β-catenin destruction complex. A TGIF-Wnt feed-forward loop is important for mammary tumorigenesis induced by hyperactivated Wnt signaling.
Transforming growth factor-β (TGF-β) regulates a wide variety of biological processes through two types of Ser/Thr transmembrane receptors: the TGF-β type I receptor and the TGF-β type II receptor ...(TβRII). Upon ligand binding, TGF-β type I receptor activated by TβRII propagates signals to Smad proteins, which mediate the activation of TGF-β target genes. In this study, we identify ADAM12 (a disintegrin and metalloproteinase 12) as a component of the TGF-β signaling pathway that acts through association with TβRII. We found that ADAM12 functions by a mechanism independent of its protease activity to facilitate the activation of TGF-β signaling, including the phosphorylation of Smad2, association of Smad2 with Smad4, and transcriptional activation. Furthermore, ADAM12 induces the accumulation of TβRII in early endosomal vesicles and stabilizes the TβRII protein presumably by suppressing the association of TβRII with Smad7. These results define ADAM12 as a new partner of TβRII that facilitates its trafficking to early endosomes in which activation of the Smad pathway is initiated.
High‐throughput screening highlighted 9‐oxo‐9H‐indeno1,2‐bpyrazine‐2,3‐dicarbonitrile (1) as an active inhibitor of ubiquitin‐specific proteases (USPs), a family of hydrolytic enzymes involved in the ...removal of ubiquitin from protein substrates. The chemical behavior of compound 1 was examined. Moreover, the synthesis and in vitro evaluation of new compounds, analogues of 1, led to the identification of potent and selective inhibitors of the deubiquitinating enzyme USP8.
Ubiquitin‐specific proteases 7 and 8 (USP7 and USP8) have been identified as promising anticancer targets. A new series of compounds that inhibit this class of cysteine proteases was discovered; selected compounds show IC50 values in the sub‐micromolar range and display in vitro specificity for USP8.
The human USP7 deubiquitinating enzyme was shown to regulate many proteins involved in the cell cycle, as well as tumor suppressors and oncogenes. Thus, USP7 offers a promising, strategic target for ...cancer therapy. Using biochemical assays and activity-based protein profiling in living systems, we identified small-molecule antagonists of USP7 and demonstrated USP7 inhibitor occupancy and selectivity in cancer cell lines. These compounds bind USP7 in the active site through a covalent mechanism. In cancer cells, these active-site-targeting inhibitors were shown to regulate the level of several USP7 substrates and thus recapitulated the USP7 knockdown phenotype that leads to G1 arrest in colon cancer cells. The data presented in this report provide proof of principle that USP7 inhibitors may be a valuable therapeutic for cancer. In addition, the discovery of such molecules offers interesting tools for studying deubiquitination.
► The USP7 deubiquitinating enzyme is a promising, strategic target for cancer therapy ► A chemical class of small molecule antagonists of USP7 was identified ► These active-site targeting inhibitors selectively occupy USP7 in living cells ► Cell proliferation and cell cycle are altered by these USP7-specific inhibitors
Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development ...of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.
Deregulation of the ubiquitin proteasome system (UPS) has been implicated in the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. The recent approval of the ...proteasome inhibitor Velcade(R) (bortezomib) for the treatment of multiple myeloma and mantle cell lymphoma establishes this system as a valid target for cancer treatment. We review here new patented proteasome inhibitors and patented small molecule inhibitors targeting more specific UPS components, such as E3 ubiquitin ligases and deubiquitylating enzymes. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).
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