We report the selection of DNA-encoded small molecule libraries against protein targets within the cytosol and on the surface of live cells. The approach relies on generation of a covalent linkage of the DNA to protein targets by affinity labeling. This cross-linking event enables subsequent copurification by a tag on the recombinant protein. To access targets within cells, a cyclic cell-penetrating peptide is appended to DNA-encoded libraries for delivery across the cell membrane. As this approach assesses binding of DELs to targets in live cells, it provides a strategy for selection of DELs against challenging targets that cannot be expressed and purified as active.
Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Towards this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumarate hydratase (FH) mutation in HLRCC cell models. Analysis of this dataset revealed an unexpected influence of local environment and pH on fumarate reactivity, and enabled the characterization of a novel a FH-regulated cysteine residue that lies at a key protein-protein interface in the SWI-SNF tumor suppressor complex. Our studies provide a powerful resource for understanding the covalent imprint of fumarate on the proteome, and lay the foundation for future efforts to exploit this distinct aspect of oncometabolism for cancer diagnosis and therapy.
Both epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are linked to metastasis via their ability to increase invasiveness and enhance tumor-initiating capacity. Growth factors, cytokines, and chemotherapies present in the tumor microenvironment (TME) are capable of inducing EMT, but the role of the extracellular matrix (ECM) in this process remains poorly understood. Here, a novel tessellated three-dimensional (3D) polymer scaffolding is used to produce a fibrillar fibronectin matrix that induces an EMT-like event that includes phosphorylation of STAT3 and requires expression of β1 integrin. Consistent with these findings, analysis of the METABRIC dataset strongly links high-level fibronectin (FN) expression to decreased patient survival. In contrast, analysis of the MCF-10A progression series indicated that intracellular FN expression was associated with nonmetastatic cells. Therefore, differential bioluminescent imaging was used to track the metastasis of isogenic epithelial and mesenchymal cells within heterogeneous tumors. Interestingly, mesenchymal tumor cells do not produce a FN matrix and cannot complete the metastatic process, even when grown within a tumor containing epithelial cells. However, mesenchymal tumor cells form FN-containing cellular fibrils capable of supporting the growth and migration of metastatic-competent tumor cells. Importantly, depletion of FN allows mesenchymal tumor cells to regain epithelial characteristics and initiate tumor growth within a metastatic microenvironment. In contrast to the tumor-promoting functions of fibronectin within the ECM, these data suggest that autocrine fibronectin production inhibits the metastatic potential of mesenchymal tumor cells. .
Metazoans contain two homologs of the Gcn5-binding protein Ada2, Ada2a and Ada2b, which nucleate formation of the ATAC and SAGA complexes, respectively. In Drosophila melanogaster, there are two splice isoforms of Ada2b: Ada2b-PA and Ada2b-PB. Here, we show that only the Ada2b-PB isoform is in SAGA; in contrast, Ada2b-PA associates with Gcn5, Ada3, Sgf29 and Chiffon, forming the Chiffon histone acetyltransferase (CHAT) complex. Chiffon is the Drosophila ortholog of Dbf4, which binds and activates the cell cycle kinase Cdc7 to initiate DNA replication. In flies, Chiffon and Cdc7 are required in ovary follicle cells for gene amplification, a specialized form of DNA re-replication. Although chiffon was previously reported to be dispensable for viability, here, we find that Chiffon is required for both histone acetylation and viability in flies. Surprisingly, we show that chiffon is a dicistronic gene that encodes distinct Cdc7-and CHATbinding polypeptides. Although the Cdc7-binding domain of Chiffon is not required for viability in flies, the CHAT-binding domain is essential for viability, but is not required for gene amplification, arguing against a role in DNA replication.
Switch/sucrose-nonfermentable (SWI/SNF) chromatin-remodeling complexes are critical regulators of chromatin dynamics during transcription, DNA replication, and DNA repair. A recently identified SWI/SNF subcomplex termed GLTSCR1/1L-BAF (GBAF; or “noncanonical BAF”, ncBAF) uniquely contains bromodomain-containing protein BRD9 and glioma tumor suppressor candidate region 1 (GLTSCR1) or its paralog GLTSCR1-like (GLTSCR1L). Recent studies have identified a unique dependency on GBAF (ncBAF) complexes in synovial sarcoma and malignant rhabdoid tumors, both of which possess aberrations in canonical BAF (cBAF) and Polybromo-BAF (PBAF) complexes. Dependencies on GBAF in malignancies without SWI/SNF aberrations, however, are less defined. Here, we show that GBAF, particularly its BRD9 subunit, is required for the viability of prostate cancer cell lines in vitro and for optimal xenograft tumor growth in vivo. BRD9 interacts with androgen receptor (AR) and CCCTC-binding factor (CTCF), and modulates AR-dependent gene expression. The GBAF complex exhibits overlapping genome localization and transcriptional targets as bromodomain and extraterminal domain–containing (BET) proteins, which are established AR coregulators. Our results demonstrate that GBAF is critical for coordinating SWI/SNF–BET cooperation and uncover a new druggable target for AR-positive prostate cancers, including those resistant to androgen deprivation or antiandrogen therapies. Significance: Advanced prostate cancers resistant to androgen receptor antagonists are still susceptible to nontoxic BRD9 inhibitors, making them a promising alternative for halting AR signaling in progressed disease.
Polycomb repressive complex 1 (PRC1) is critical for mediating gene repression during development and adult stem cell maintenance. Five CBX proteins, CBX2,4,6,7,8, form mutually exclusive PRC1 complexes and are thought to play a role in the association of PRC1 with chromatin. Specifically, the N-terminal chromodomain (CD) in the CBX proteins is thought to mediate specific targeting to methylated histones. For CBX8, however, the chromodomain has demonstrated weak affinity and specificity for methylated histones in vitro, leaving doubt as to its role in CBX8 chromatin association. Here, we investigate the function of the CBX8 CD in vitro and in vivo. We find that the CD is in fact a major driver of CBX8 chromatin association and determine that this is driven by both histone and previously unrecognized DNA binding activity. We characterize the structural basis of histone and DNA binding and determine how they integrate on multiple levels. Notably, we find that the chromatin environment is critical in determining the ultimate function of the CD in CBX8 association.
Functional characterization of adenylyl cyclase (AC) isoforms has proven challenging in mammalian cells because of the endogenous expression of multiple AC isoforms and the high background cAMP levels induced by nonselective AC activators. To simplify the characterization of individual transmembrane AC (mAC) isoforms, we generated a human embryonic kidney cell line 293 (HEK293) with low cAMP levels by knocking out two highly expressed ACs, AC3 and AC6, using CRISPR/Cas9 technology. Stable HEK293 cell lines lacking either AC6 (HEK-ACΔ6) or both AC3 and AC6 (HEK-ACΔ3/6) were generated. Knockout was confirmed genetically and by comparing cAMP responses of the knockout cells to the parental cell line. HEK-ACΔ6 and HEK-ACΔ3/6 cells revealed an 85% and 95% reduction in the forskolin-stimulated cAMP response, respectively. Forskolin- and G-coupled receptor-induced activation was examined for the nine recombinant mAC isoforms in the HEK-ACΔ3/6 cells. Forskolin-mediated cAMP accumulation for AC1-6 and AC8 revealed 10- to 250-fold increases over the basal cAMP levels. All nine mAC isoforms, except AC8, also exhibited significantly higher cAMP levels than the control cells after G-coupled receptor activation. Isoform-specific AC regulation by protein kinases and Ca/calmodulin was also recapitulated in the knockout cells. Furthermore, the utility of the HEK-ACΔ3/6 cell line was demonstrated by characterizing the activity of novel AC1 forskolin binding-site mutants. Hence, we have developed a HEK293 cell line deficient of endogenous AC3 and AC6 with low cAMP background levels for studies of cAMP signaling and AC isoform regulation.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.