Person:

King, Randall

Because off-target effects hamper interpretation and validation of RNAi screens, we developed a bioinformatics method, Genome-wide Enrichment of Seed Sequence matches (GESS), to identify candidate off-targeted transcripts from direct analysis of primary screening data. GESS identified a prominent off-targeted transcript in several screens, including MAD2 in a screen for components of the spindle assembly checkpoint. We demonstrate how incorporation of the results of GESS analysis can enhance the validation rate in RNAi screens.

The Anaphase-Promoting Complex/Cyclosome (APC) is a ubiquitin ligase required for exit from mitosis. We previously showed that Tosyl Arginine Methyl Ester (TAME) inhibits APC-dependent proteolysis by competing with the C-terminal IR-tail of the APC activator Cdc20 for APC binding. Here we show that in the absence of APC substrates, TAME ejects Cdc20 from the APC by promoting Cdc20 auto-ubiquitination in its N-terminal region. Cyclin B1 antagonizes TAME's effect by promoting binding of free Cdc20 to the APC and suppressing Cdc20 auto-ubiquitination. Nevertheless, TAME stabilizes cyclin B1 in Xenopus extract by two mechanisms. First, it reduces the (k_{cat}) of the (APC^{Cdc20})/cyclin B1 complex without affecting the (K_m), slowing the initial ubiquitination of unmodified cyclin B1. Second, as cyclin B1 becomes ubiquitinated, it loses its ability to promote Cdc20 binding to the APC in the presence of TAME. As a result, cyclin B1 ubiquitination terminates before reaching the threshold necessary for proteolysis.

Cyclin B1–CDK1 activity is essential for mitotic entry, but questions remain regarding how the activity of this kinase is spatially regulated. Previous studies showed that the cyclin B1 subunit localizes to several compartments of a mitotic cell, including the centrosomes, mitotic spindle, kinetochores and chromosomes via distinct sequence elements. Mitotic chromosome association occurs through the unstructured N-terminal domain of cyclin B1 and is independent of CDK1 binding. Here, we use live cell imaging of human cyclin B1 fused to GFP to precisely define the sequence elements within cyclin B1 that mediate its association with condensed mitotic chromosomes. We find that a short, evolutionarily conserved N-terminal motif is required for cyclin B1 to localize to mitotic chromosomes. We further reveal a role for arginine residues within and near the destruction box sequence in the chromosome association of cyclin B1. Additionally, our data suggest that sequences further downstream in cyclin B1, such as the cytoplasmic retention sequence and the cyclin box, may negatively modulate chromosome association. Because multiple basic residues are required for cyclin B1 association with mitotic chromosomes, electrostatic interactions with DNA may facilitate cyclin B1 localization to chromosomes.

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes1-9. Its effects on protein turnover are substrate specific, for unknown reasons. We report that USP14 shows a dramatic preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14’s catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin chain architecture.

Conventional high-throughput ‘chemical genetic’ screening seeks to identify small-molecule inhibitors of a specific protein or pathway. A recent study describes how unbiased screening of cellular morphology, followed by affinity purification to identify targets of compounds with interesting effects, can lead to the identification of novel inhibitors.

Small-molecule antagonists of Hedgehog-pathway signaling, such as cyclopamine, have been known for some time. Now, small-molecule agonists of the Hedgehog pathway have also been identified. The finding that both antagonists and agonists target the protein Smoothened supports the emerging hypothesis that Smoothened may be regulated by endogenous small molecules.

The anaphase promoting complex/cyclosome (APC/C) is an ubiquitin ligase involved in cell cycle. During the metaphase-anaphase transition the APC/C is activated by Cdc20. The aim of this study is to elucidate the importance and therapeutic potential of APC/C and its co-activator Cdc20 in multiple myeloma (MM). Gene expression analysis revealed that Cdc20 was expressed at higher levels in gene expression-based high-risk MM patients. Moreover, high Cdc20 expression correlated with poor prognosis. Treatment of human myeloma cell lines with proTAME, an APC/C inhibitor, resulted in an accumulation of APC/CCdc20 substrate cyclin B1 and an accumulation of cells in metaphase. Moreover we observed a significant dose-dependent decrease in viability and increase in apoptosis in MM cells upon proTAME treatment. The induction of apoptosis was accompanied with caspase 3, 8, 9 and PARP cleavage. A similar metaphase arrest and induction of apoptosis were obtained with specific knockdown of Cdc20. In addition, we demonstrated the accumulation of Bim was partially responsible for the observed cell death. Combining proTAME with another APC/C inhibitor apcin or the alkylating agent melphalan resulted in enhanced anti-MM activity. This study suggests that the APC/C and its co-activator Cdc20 could be a new and promising target especially in high-risk MM patients.

Warsaw breakage syndrome (WABS) is caused by defective DDX11, a DNA helicase that is essential for chromatid cohesion. Here, a paired genome-wide siRNA screen in patient-derived cell lines reveals that WABS cells do not tolerate partial depletion of individual APC/C subunits or the spindle checkpoint inhibitor p31comet. A combination of reduced cohesion and impaired APC/C function also leads to fatal mitotic arrest in diploid RPE1 cells. Moreover, WABS cell lines, and several cancer cell lines with cohesion defects, display a highly increased response to a new cell-permeable APC/C inhibitor, apcin, but not to the spindle poison paclitaxel. Synthetic lethality of APC/C inhibition and cohesion defects strictly depends on a functional mitotic spindle checkpoint as well as on intact microtubule pulling forces. This indicates that the underlying mechanism involves cohesion fatigue in response to mitotic delay, leading to spindle checkpoint re-activation and lethal mitotic arrest. Our results point to APC/C inhibitors as promising therapeutic agents targeting cohesion-defective cancers.