Active growth signaling promotes senescence and cancer cell sensitivity to CDK7 inhibition.

Tumor growth is driven by continued cellular growth and proliferation. Cyclin-dependent kinase 7's (CDK7) role in activating mitotic CDKs and global gene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here, we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell-cycle exit, known as senescence, without promoting DNA damage signaling or cell death. A chemogenetic genome-wide CRISPR knockout screen identified that active mTOR (mammalian target of rapamycin) signaling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signaling correlates with sensitivity in cancer cell lines. Reverting a growth-promoting mutation in PIK3CA to wild type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.

Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells.

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.

In the ubiquitin-proteasome system (UPS), E2 enzymes mediate the conjugation of ubiquitin to substrates and thereby control protein stability and interactions. The E2 enzyme hCdc34 catalyzes the ubiquitination of hundreds of proteins in conjunction with the cullin-RING (CRL) superfamily of E3 enzymes. We identified a small molecule termed CC0651 that selectively inhibits hCdc34. Structure determination revealed that CC0651 inserts into a cryptic binding pocket on hCdc34 distant from the catalytic site, causing subtle but wholesale displacement of E2 secondary structural elements. CC0651 analogs inhibited proliferation of human cancer cell lines and caused accumulation of the SCF(Skp2) substrate p27(Kip1). CC0651 does not affect hCdc34 interactions with E1 or E3 enzymes or the formation of the ubiquitin thioester but instead interferes with the discharge of ubiquitin to acceptor lysine residues. E2 enzymes are thus susceptible to noncatalytic site inhibition and may represent a viable class of drug target in the UPS.

At Long Last, a C-Terminal Bookend for the Ubiquitin Code.

The ubiquitin-proteasome system controls the stability of myriad protein substrates via short sequence motifs called degrons. Studies by Koren et al. (2018) and Lin et al. (2018) have uncovered a broad new class of degrons located at the extreme C terminus of substrates.

Cdc48/VCP Promotes Chromosome Morphogenesis by Releasing Condensin from Self-Entrapment in Chromatin.

The morphological transformation of amorphous chromatin into distinct chromosomes is a hallmark of mitosis. To achieve this, chromatin must be compacted and remodeled by a ring-shaped enzyme complex known as condensin. However, the mechanistic basis underpinning condensin's role in chromosome remodeling has remained elusive. Here we show that condensin has a strong tendency to trap itself in its own reaction product during chromatin compaction and yet is capable of interacting with chromatin in a highly dynamic manner in vivo. To resolve this apparent paradox, we identified specific chromatin remodelers and AAA-class ATPases that act in a coordinated manner to release condensin from chromatin entrapment. The Cdc48 segregase is the central linchpin of this regulatory mechanism and promotes ubiquitin-dependent cycling of condensin on mitotic chromatin as well as effective chromosome condensation. Collectively, our results show that condensin inhibition by its own reaction product is relieved by forceful enzyme extraction from chromatin.

Genetic enhancers of partial PLK1 inhibition reveal hypersensitivity to kinetochore perturbations.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors.

The microprotein Nrs1 rewires the G1/S transcriptional machinery during nitrogen limitation in budding yeast.

Commitment to cell division at the end of G1 phase, termed Start in the budding yeast Saccharomyces cerevisiae, is strongly influenced by nutrient availability. To identify new dominant activators of Start that might operate under different nutrient conditions, we screened a genome-wide ORF overexpression library for genes that bypass a Start arrest caused by absence of the G1 cyclin Cln3 and the transcriptional activator Bck2. We recovered a hypothetical gene YLR053c, renamed NRS1 for Nitrogen-Responsive Start regulator 1, which encodes a poorly characterized 108 amino acid microprotein. Endogenous Nrs1 was nuclear-localized, restricted to poor nitrogen conditions, induced upon TORC1 inhibition, and cell cycle-regulated with a peak at Start. NRS1 interacted genetically with SWI4 and SWI6, which encode subunits of the main G1/S transcription factor complex SBF. Correspondingly, Nrs1 physically interacted with Swi4 and Swi6 and was localized to G1/S promoter DNA. Nrs1 exhibited inherent transactivation activity, and fusion of Nrs1 to the SBF inhibitor Whi5 was sufficient to suppress other Start defects. Nrs1 appears to be a recently evolved microprotein that rewires the G1/S transcriptional machinery under poor nitrogen conditions.

Synthetic Genomics: Rewriting the Genome Chromosome by Chromosome.

The Synthetic Yeast Genome Project (Sc2.0) consortium has recently published a collection of seven reports on the design, assembly, and in vivo characterization of five entirely synthetic yeast chromosomes that set the stage for de novo construction of synthetic custom-engineered eukaryotes.

The p38/HOG stress-activated protein kinase network couples growth to division in Candida albicans.

Cell size is a complex trait that responds to developmental and environmental cues. Quantitative size analysis of mutant strain collections disrupted for protein kinases and transcriptional regulators in the pathogenic yeast Candida albicans uncovered 66 genes that altered cell size, few of which overlapped with known size genes in the budding yeast Saccharomyces cerevisiae. A potent size regulator specific to C. albicans was the conserved p38/HOG MAPK module that mediates the osmostress response. Basal HOG activity inhibited the SBF G1/S transcription factor complex in a stress-independent fashion to delay the G1/S transition. The HOG network also governed ribosome biogenesis through the master transcriptional regulator Sfp1. Hog1 bound to the promoters and cognate transcription factors for ribosome biogenesis regulons and interacted genetically with the SBF G1/S machinery, and thereby directly linked cell growth and division. These results illuminate the evolutionary plasticity of size control and identify the HOG module as a nexus of cell cycle and growth regulation.

Condensin ATPase motifs contribute differentially to the maintenance of chromosome morphology and genome stability.

Effective transfer of genetic information during cell division requires a major reorganization of chromosome structure. This process is triggered by condensin, a conserved pentameric ATPase essential for chromosome condensation. How condensin harnesses the energy of ATP hydrolysis to promote chromatin reorganization is unknown. To address this issue, we performed a genetic screen specifically focused on the ATPase domain of Smc4, a core subunit of condensin. Our screen identified mutational hotspots that impair condensin's ability to condense chromosomes to various degrees. These mutations have distinct effects on viability, genome stability, and chromosome morphology, revealing unique thresholds for condensin enzymatic activity in the execution of its cellular functions. Biochemical analyses indicate that inactivation of Smc4 ATPase activity can result in cell lethality because it favors a specific configuration of condensin that locks ATP in the enzyme. Together, our results provide critical insights into the mechanism used by condensin to harness the energy of ATP hydrolysis for the compaction of chromatin.

The BioGRID interaction database: 2019 update.

The Biological General Repository for Interaction Datasets (BioGRID: https://thebiogrid.org) is an open access database dedicated to the curation and archival storage of protein, genetic and chemical interactions for all major model organism species and humans. As of September 2018 (build 3.4.164), BioGRID contains records for 1 598 688 biological interactions manually annotated from 55 809 publications for 71 species, as classified by an updated set of controlled vocabularies for experimental detection methods. BioGRID also houses records for >700 000 post-translational modification sites. BioGRID now captures chemical interaction data, including chemical-protein interactions for human drug targets drawn from the DrugBank database and manually curated bioactive compounds reported in the literature. A new dedicated aspect of BioGRID annotates genome-wide CRISPR/Cas9-based screens that report gene-phenotype and gene-gene relationships. An extension of the BioGRID resource called the Open Repository for CRISPR Screens (ORCS) database (https://orcs.thebiogrid.org) currently contains over 500 genome-wide screens carried out in human or mouse cell lines. All data in BioGRID is made freely available without restriction, is directly downloadable in standard formats and can be readily incorporated into existing applications via our web service platforms. BioGRID data are also freely distributed through partner model organism databases and meta-databases.

Proteome-wide, Structure-Based Prediction of Protein-Protein Interactions/New Molecular Interactions Viewer.

Determining the complete Arabidopsis (Arabidopsis thaliana) protein-protein interaction network is essential for understanding the functional organization of the proteome. Numerous small-scale studies and a couple of large-scale ones have elucidated a fraction of the estimated 300,000 binary protein-protein interactions in Arabidopsis. In this study, we provide evidence that a docking algorithm has the ability to identify real interactions using both experimentally determined and predicted protein structures. We ranked 0.91 million interactions generated by all possible pairwise combinations of 1,346 predicted structure models from an Arabidopsis predicted "structure-ome" and found a significant enrichment of real interactions for the top-ranking predicted interactions, as shown by cosubcellular enrichment analysis and yeast two-hybrid validation. Our success rate for computationally predicted, structure-based interactions was 63% of the success rate for published interactions naively tested using the yeast two-hybrid system and 2.7 times better than for randomly picked pairs of proteins. This study provides another perspective in interactome exploration and biological network reconstruction using protein structural information. We have made these interactions freely accessible through an improved Arabidopsis Interactions Viewer and have created community tools for accessing these and ∼2.8 million other protein-protein and protein-DNA interactions for hypothesis generation by researchers worldwide. The Arabidopsis Interactions Viewer is freely available at http://bar.utoronto.ca/interactions2/.

Machine Learning of Global Phosphoproteomic Profiles Enables Discrimination of Direct versus Indirect Kinase Substrates.

Mass spectrometry allows quantification of tens of thousands of phosphorylation sites from minute amounts of cellular material. Despite this wealth of information, our understanding of phosphorylation-based signaling is limited, in part because it is not possible to deconvolute substrate phosphorylation that is directly mediated by a particular kinase versus phosphorylation that is mediated by downstream kinases. Here, we describe a framework for assignment of direct in vivo kinase substrates using a combination of selective chemical inhibition, quantitative phosphoproteomics, and machine learning techniques. Our workflow allows classification of phosphorylation events following inhibition of an analog-sensitive kinase into kinase-independent effects of the inhibitor, direct effects on cognate substrates, and indirect effects mediated by downstream kinases or phosphatases. We applied this method to identify many direct targets of Cdc28 and Snf1 kinases in the budding yeast Saccharomyces cerevisiae Global phosphoproteome analysis of acute time-series demonstrated that dephosphorylation of direct kinase substrates occurs more rapidly compared with indirect substrates, both after inhibitor treatment and under a physiological nutrient shift in wt cells. Mutagenesis experiments revealed a high proportion of functionally relevant phosphorylation sites on Snf1 targets. For example, Snf1 itself was inhibited through autophosphorylation on Ser391 and new phosphosites were discovered that modulate the activity of the Reg1 regulatory subunit of the Glc7 phosphatase and the Gal83 ß-subunit of SNF1 complex. This methodology applies to any kinase for which a functional analog sensitive version can be constructed to facilitate the dissection of the global phosphorylation network.

The BioGRID interaction database: 2017 update.

The Biological General Repository for Interaction Datasets (BioGRID: https://thebiogrid.org) is an open access database dedicated to the annotation and archival of protein, genetic and chemical interactions for all major model organism species and humans. As of September 2016 (build 3.4.140), the BioGRID contains 1 072 173 genetic and protein interactions, and 38 559 post-translational modifications, as manually annotated from 48 114 publications. This dataset represents interaction records for 66 model organisms and represents a 30% increase compared to the previous 2015 BioGRID update. BioGRID curates the biomedical literature for major model organism species, including humans, with a recent emphasis on central biological processes and specific human diseases. To facilitate network-based approaches to drug discovery, BioGRID now incorporates 27 501 chemical-protein interactions for human drug targets, as drawn from the DrugBank database. A new dynamic interaction network viewer allows the easy navigation and filtering of all genetic and protein interaction data, as well as for bioactive compounds and their established targets. BioGRID data are directly downloadable without restriction in a variety of standardized formats and are freely distributed through partner model organism databases and meta-databases.

Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1-F-box interface.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

Real-time tracking of complex ubiquitination cascades using a fluorescent confocal on-bead assay.

BACKGROUND: The ubiquitin-proteasome system (UPS) controls the stability, localization and/or activity of the proteome. However, the identification and characterization of complex individual ubiquitination cascades and their modulators remains a challenge. Here, we report a broadly applicable, multiplexed, miniaturized on-bead technique for real-time monitoring of various ubiquitination-related enzymatic activities. The assay, termed UPS-confocal fluorescence nanoscanning (UPS-CONA), employs a substrate of interest immobilized on a micro-bead and a fluorescently labeled ubiquitin which, upon enzymatic conjugation to the substrate, is quantitatively detected on the bead periphery by confocal imaging. RESULTS: UPS-CONA is suitable for studying individual enzymatic activities, including various E1, E2, and HECT-type E3 enzymes, and for monitoring multi-step reactions within ubiquitination cascades in a single experimental compartment. We demonstrate the power of the UPS-CONA technique by simultaneously following ubiquitin transfer from Ube1 through Ube2L3 to E6AP. We applied this multi-step setup to investigate the selectivity of five ubiquitination inhibitors reportedly targeting different classes of ubiquitination enzymes. Using UPS-CONA, we have identified a new activity of a small molecule E2 inhibitor, BAY 11-7082, and of a HECT E3 inhibitor, heclin, towards the Ube1 enzyme. CONCLUSIONS: As a sensitive, quantitative, flexible, and reagent-efficient method with a straightforward protocol, UPS-CONA constitutes a powerful tool for interrogation of ubiquitination-related enzymatic pathways and their chemical modulators, and is readily scalable for large experiments.

Combined Enrichment/Enzymatic Approach To Study Tightly Clustered Multisite Phosphorylation on Ser-Rich Domains.

The regulation of protein function through phosphorylation is often dominated by allosteric interactions and conformational changes. However, alternative mechanisms involving electrostatic interactions also regulate protein function. In particular, phosphorylation of clusters of Ser/Thr residues can affect protein-plasma membrane/chromatin interactions by electrostatic interactions between phosphosites and phospholipids or histones. Currently, only a few examples of such mechanisms are reported, primarily because of the difficulties of detecting highly phosphorylated proteins and peptides, due in part to the low ionization efficiency and fragmentation yield of multiphosphorylated peptides in mass spectrometry when using positive ion mode detection. This difficulty in detection has resulted in under-reporting of such modified regions, which can be thought of as phosphoproteomic dark matter. Here, we present a novel approach that enriches for multisite-phosphorylated peptides that until now remained inaccessible by conventional phosphoproteomics. Our technique enables the identification of multisite-phosphorylated regions on more than 300 proteins in both yeast and human cells and can be used to profile changes in multisite phosphorylation upon cell stimulation. We further characterize the role of multisite phosphorylation for Ste20 in the yeast mating pheromone response. Mutagenesis experiments confirmed that multisite phosphorylation of Ser/Thr-rich regions plays an important role in the regulation of Ste20 activity during mating pheromone signaling. The ability to detect protein multisite phosphorylation opens new avenues to explore phosphoproteomic dark matter and to study Ser-rich proteins that interact with binding partners through charge pairing mechanisms.

CLMSVault: A Software Suite for Protein Cross-Linking Mass-Spectrometry Data Analysis and Visualization.

Protein cross-linking mass spectrometry (CL-MS) enables the sensitive detection of protein interactions and the inference of protein complex topology. The detection of chemical cross-links between protein residues can identify intra- and interprotein contact sites or provide physical constraints for molecular modeling of protein structure. Recent innovations in cross-linker design, sample preparation, mass spectrometry, and software tools have significantly improved CL-MS approaches. Although a number of algorithms now exist for the identification of cross-linked peptides from mass spectral data, a dearth of user-friendly analysis tools represent a practical bottleneck to the broad adoption of the approach. To facilitate the analysis of CL-MS data, we developed CLMSVault, a software suite designed to leverage existing CL-MS algorithms and provide intuitive and flexible tools for cross-platform data interpretation. CLMSVault stores and combines complementary information obtained from different cross-linkers and search algorithms. CLMSVault provides filtering, comparison, and visualization tools to support CL-MS analyses and includes a workflow for label-free quantification of cross-linked peptides. An embedded 3D viewer enables the visualization of quantitative data and the mapping of cross-linked sites onto PDB structural models. We demonstrate the application of CLMSVault for the analysis of a noncovalent Cdc34-ubiquitin protein complex cross-linked under different conditions. CLMSVault is open-source software (available at https://gitlab.com/courcelm/clmsvault.git ), and a live demo is available at http://democlmsvault.tyerslab.com/ .

The BioGRID interaction database: 2015 update.

The Biological General Repository for Interaction Datasets (BioGRID: http://thebiogrid.org) is an open access database that houses genetic and protein interactions curated from the primary biomedical literature for all major model organism species and humans. As of September 2014, the BioGRID contains 749,912 interactions as drawn from 43,149 publications that represent 30 model organisms. This interaction count represents a 50% increase compared to our previous 2013 BioGRID update. BioGRID data are freely distributed through partner model organism databases and meta-databases and are directly downloadable in a variety of formats. In addition to general curation of the published literature for the major model species, BioGRID undertakes themed curation projects in areas of particular relevance for biomedical sciences, such as the ubiquitin-proteasome system and various human disease-associated interaction networks. BioGRID curation is coordinated through an Interaction Management System (IMS) that facilitates the compilation interaction records through structured evidence codes, phenotype ontologies, and gene annotation. The BioGRID architecture has been improved in order to support a broader range of interaction and post-translational modification types, to allow the representation of more complex multi-gene/protein interactions, to account for cellular phenotypes through structured ontologies, to expedite curation through semi-automated text-mining approaches, and to enhance curation quality control.