Stuck in the middle: drugging the ubiquitin system at the e2 step.

The discovery of a small-molecule allosteric inhibitor of the CDC34 ubiquitin-conjugating enzyme (E2) by Ceccarelli et al. raises the possibility that it will be generally feasible to selectively inhibit ubiquitin transfer at this central step in the ubiquitin pathway.

Using cognitive load theory to evaluate and improve preparatory materials and study time for the flipped classroom.

BACKGROUND: Preclinical medical education is content-dense and time-constrained. Flipped classroom approaches promote durable learning, but challenges with unsatisfactory student preparation and high workload remain. Cognitive load theory defines instructional design as "efficient" if learners can master the presented concepts without cognitive overload. We created a PReparatory Evaluation Process (PREP) to systematically assess and measure improvement in the cognitive-load efficiency of preparatory materials and impact on study time (time-efficiency). METHODS: We conducted this study in a flipped, multidisciplinary course for ~ 170 first year students at Harvard Medical School using a naturalistic post-test design. For each flipped session (n = 97), we assessed cognitive load and preparatory study time by administering a 3-item PREP survey embedded within a short subject-matter quiz students completed before class. Over three years (2017-2019), we evaluated cognitive load- and time- based efficiency to guide iterative revisions of the materials by content experts. The ability of PREP to detect changes to the instructional design (sensitivity) was validated through a manual audit of the materials. RESULTS: The average survey response rate was ≥ 94%. Content expertise was not required to interpret PREP data. Initially students did not necessarily allocate the most study time to the most difficult content. Over time, the iterative changes in instructional design increased the cognitive load- and time-based efficiency of preparatory materials with large effect sizes (p < .01). Furthermore, this increased the overall alignment of cognitive load with study time: students allocated more time to difficult content away from more familiar, less difficult content without increasing workload overall. CONCLUSIONS: Cognitive load and time constraints are important parameters to consider when designing curricula. The PREP process is learner-centered, grounded in educational theory, and works independently of content knowledge. It can provide rich and actionable insights into instructional design of flipped classes not captured by traditional satisfaction-based evaluations.

Paradoxical mitotic exit induced by a small molecule inhibitor of APC/CCdc20.

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase that initiates anaphase and mitotic exit. APC/C is activated by Cdc20 and inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin as a small molecule ligand of Cdc20 that inhibits APC/CCdc20 and prolongs mitosis. Here we find that apcin paradoxically shortens mitosis when SAC activity is high. These opposing effects of apcin arise from targeting of a common binding site in Cdc20 required for both substrate ubiquitination and MCC-dependent APC/C inhibition. Furthermore, we found that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin therefore causes either net APC/C inhibition, prolonging mitosis when SAC activity is low, or net APC/C activation, shortening mitosis when SAC activity is high, demonstrating that a small molecule can produce opposing biological effects depending on regulatory context.

USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites.

USP14 is a major regulator of the proteasome and one of three proteasome-associated deubiquitinating enzymes. Its effects on protein turnover are substrate-specific, for unknown reasons. We report that USP14 shows a marked 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 conjugate architecture.

The Insulin Receptor Adaptor IRS2 is an APC/C Substrate That Promotes Cell Cycle Protein Expression and a Robust Spindle Assembly Checkpoint.

Insulin receptor substrate 2 (IRS2) is an essential adaptor that mediates signaling downstream of the insulin receptor and other receptor tyrosine kinases. Transduction through IRS2-dependent pathways is important for coordinating metabolic homeostasis, and dysregulation of IRS2 causes systemic insulin signaling defects. Despite the importance of maintaining proper IRS2 abundance, little is known about what factors mediate its protein stability. We conducted an unbiased proteomic screen to uncover novel substrates of the Anaphase Promoting Complex/Cyclosome (APC/C), a ubiquitin ligase that controls the abundance of key cell cycle regulators. We found that IRS2 levels are regulated by APC/C activity and that IRS2 is a direct APC/C target in G1 Consistent with the APC/C's role in degrading cell cycle regulators, quantitative proteomic analysis of IRS2-null cells revealed a deficiency in proteins involved in cell cycle progression. We further show that cells lacking IRS2 display a weakened spindle assembly checkpoint in cells treated with microtubule inhibitors. Together, these findings reveal a new pathway for IRS2 turnover and indicate that IRS2 is a component of the cell cycle control system in addition to acting as an essential metabolic regulator.

Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C.

Protein machines are multi-subunit protein complexes that orchestrate highly regulated biochemical tasks. An example is the anaphase-promoting complex/cyclosome (APC/C), a 13-subunit ubiquitin ligase that initiates the metaphase-anaphase transition and mitotic exit by targeting proteins such as securin and cyclin B1 for ubiquitin-dependent destruction by the proteasome. Because blocking mitotic exit is an effective approach for inducing tumour cell death, the APC/C represents a potential novel target for cancer therapy. APC/C activation in mitosis requires binding of Cdc20 (ref. 5), which forms a co-receptor with the APC/C to recognize substrates containing a destruction box (D-box). Here we demonstrate that we can synergistically inhibit APC/C-dependent proteolysis and mitotic exit by simultaneously disrupting two protein-protein interactions within the APC/C-Cdc20-substrate ternary complex. We identify a small molecule, called apcin (APC inhibitor), which binds to Cdc20 and competitively inhibits the ubiquitylation of D-box-containing substrates. Analysis of the crystal structure of the apcin-Cdc20 complex suggests that apcin occupies the D-box-binding pocket on the side face of the WD40-domain. The ability of apcin to block mitotic exit is synergistically amplified by co-addition of tosyl-l-arginine methyl ester, a small molecule that blocks the APC/C-Cdc20 interaction. This work suggests that simultaneous disruption of multiple, weak protein-protein interactions is an effective approach for inactivating a protein machine.

An inhibitor of the proteasomal deubiquitinating enzyme USP14 induces tau elimination in cultured neurons.

The ubiquitin-proteasome system (UPS) is responsible for most selective protein degradation in eukaryotes and regulates numerous cellular processes, including cell cycle control and protein quality control. A component of this system, the deubiquitinating enzyme USP14, associates with the proteasome where it can rescue substrates from degradation by removal of the ubiquitin tag. We previously found that a small-molecule inhibitor of USP14, known as IU1, can increase the rate of degradation of a subset of proteasome substrates. We report here the synthesis and characterization of 87 variants of IU1, which resulted in the identification of a 10-fold more potent USP14 inhibitor that retains specificity for USP14. The capacity of this compound, IU1-47, to enhance protein degradation in cells was tested using as a reporter the microtubule-associated protein tau, which has been implicated in many neurodegenerative diseases. Using primary neuronal cultures, IU1-47 was found to accelerate the rate of degradation of wild-type tau, the pathological tau mutants P301L and P301S, and the A152T tau variant. We also report that a specific residue in tau, lysine 174, is critical for the IU1-47-mediated tau degradation by the proteasome. Finally, we show that IU1-47 stimulates autophagic flux in primary neurons. In summary, these findings provide a powerful research tool for investigating the complex biology of USP14.

Enhancement of proteasome activity by a small-molecule inhibitor of USP14.

Proteasomes, the primary mediators of ubiquitin-protein conjugate degradation, are regulated through complex and poorly understood mechanisms. Here we show that USP14, a proteasome-associated deubiquitinating enzyme, can inhibit the degradation of ubiquitin-protein conjugates both in vitro and in cells. A catalytically inactive variant of USP14 has reduced inhibitory activity, indicating that inhibition is mediated by trimming of the ubiquitin chain on the substrate. A high-throughput screen identified a selective small-molecule inhibitor of the deubiquitinating activity of human USP14. Treatment of cultured cells with this compound enhanced degradation of several proteasome substrates that have been implicated in neurodegenerative disease. USP14 inhibition accelerated the degradation of oxidized proteins and enhanced resistance to oxidative stress. Enhancement of proteasome activity through inhibition of USP14 may offer a strategy to reduce the levels of aberrant proteins in cells under proteotoxic stress.

A bioinformatics method identifies prominent off-targeted transcripts in RNAi screens.

Because off-target effects hamper interpretation and validation of RNAi screen data, we developed a bioinformatics method, genome-wide enrichment of seed sequence matches (GESS), to identify candidate off-targeted transcripts in primary screening data. GESS analysis revealed a prominent off-targeted transcript in several screens, including MAD2 (MAD2L1) in a screen for genes required for the spindle assembly checkpoint. GESS analysis results can enhance the validation rate in RNAi screens.

An APC/C inhibitor stabilizes cyclin B1 by prematurely terminating ubiquitination.

The anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that is required for exit from mitosis. We previously showed that tosyl arginine methyl ester (TAME) inhibits APC-dependent proteolysis by competing with the C-terminal isoleucine-arginine tail of the APC activator cell division cycle 20 (Cdc20) for APC binding. Here we show that in the absence of APC substrates, TAME ejects Cdc20 from the APC by promoting Cdc20 autoubiquitination in its N-terminal region. Cyclin B1 antagonizes TAME's effect by promoting binding of free Cdc20 to the APC and by suppressing Cdc20 autoubiquitination. Nevertheless, TAME stabilizes cyclin B1 in Xenopus extracts 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.

Specificity profiling of deubiquitylases against endogenously generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high-impact DUBs (USP7, USP9X, USP36, USP15, and USP24) that each reduced ubiquitylation of over 10% of the isolated proteins. Candidate substrates of high-impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology.

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.

Trimming of ubiquitin chains by proteasome-associated deubiquitinating enzymes.

The proteasome generally recognizes substrate via its multiubiquitin chain followed by ATP-dependent unfolding and translocation of the substrate from the regulatory particle into the proteolytic core particle to be degraded. Substrate-bound ubiquitin groups are for the most part not delivered to the core particle and broken down together with substrate but instead recovered as intact free ubiquitin and ubiquitin chains. Substrate deubiquitination on the proteasome is mediated by three distinct deubiquitinating enzymes associated with the regulatory particle: RPN11, UCH37, and USP14. RPN11 cleaves at the base of the ubiquitin chain where it is linked to the substrate, whereas UCH37 and apparently USP14 mediate a stepwise removal of ubiquitin from the substrate by disassembling the chain from its distal tip. In contrast to UCH37 and USP14, RPN11 shows degradation-coupled activity; RPN11-mediated deubiquitination is apparently delayed until the proteasome is committed to degrade the substrate. Accordingly, RPN11-mediated deubiquitination promotes substrate degradation. In contrast, removal of ubiquitin prior to commitment could antagonize substrate degradation by promoting substrate dissociation from the proteasome. Emerging evidence suggests that USP14 and UCH37 can both suppress substrate degradation in this way. One line of study has shown that small molecule USP14 inhibitors can enhance proteasome function in cells, which is consistent with this model. Enhancing protein degradation could potentially have therapeutic applications for diseases involving toxic proteins that are proteasome substrates. However, the responsiveness of substrates to inhibition of proteasomal deubiquitinating enzymes may vary substantially. This substrate specificity and its mechanistic basis should be addressed in future studies.

Identification of Deubiquitinase Substrates in Xenopus Egg Extract.

Deubiquitinases (DUBs) antagonize protein ubiquitination by removing ubiquitin from substrates. Identifying the physiological substrates of each DUB is critical for understanding DUB function and the principles that govern the specificity of this class of enzymes. Since multiple DUBs can act on the same substrate, it can be challenging to identify substrates using inactivating a single enzyme. Here, we outline a method that enables the identification of proteins whose stability depends on DUB activity and an approach to profile DUB specificity in Xenopus egg extract. By coupling broad DUB inhibition with quantitative proteomics, we circumvent DUB redundancy to identify DUB substrates. By adding back recombinant DUBs individually to the extract, we pinpoint DUBs sufficient to counteract proteasomal degradation of these newly identified substrates. We apply this method to Xenopus egg extract but suggest that it can also be adapted to other cell lysates.

Substrate identification and specificity profiling of deubiquitylases against endogenously-generated ubiquitin-protein conjugates.

Deubiquitylating enzymes (DUBs) remove ubiquitin from proteins thereby regulating their stability or activity. Our understanding of DUB-substrate specificity is limited because DUBs are typically not compared to each other against many physiological substrates. By broadly inhibiting DUBs in Xenopus egg extract, we generated hundreds of ubiquitylated proteins and compared the ability of 30 DUBs to deubiquitylate them using quantitative proteomics. We identified five high impact DUBs (USP7, USP9X, USP36, USP15 and USP24) that each reduced ubiquitylation of over ten percent of the isolated proteins. Candidate substrates of high impact DUBs showed substantial overlap and were enriched for disordered regions, suggesting this feature may promote substrate recognition. Other DUBs showed lower impact and non-overlapping specificity, targeting distinct non-disordered proteins including complexes such as the ribosome or the proteasome. Altogether our study identifies candidate DUB substrates and defines patterns of functional redundancy and specificity, revealing substrate characteristics that may influence DUB-substrate recognition.

Dissecting cell biology with chemical scalpels.

Understanding complex cellular processes requires methods for specifically perturbing protein function in a temporally defined fashion. In recent years a variety of chemical tools have been used to study the cytoskeleton and cell division, protein trafficking, and the destruction of proteins by the ubiquitin-proteasome system. The ability to use combinations of reversible inhibitors is proving to be especially helpful in dissecting complex cellular events. Furthermore, the identification of novel inhibitors through unbiased screening approaches is revealing novel drugable steps in important cellular pathways.

Advanced Integrated Science Courses: Building a Skill Set to Engage With the Interface of Research and Medicine.

Scientific research has been changing medical practice at an increasing pace. To keep up with this change, physicians of the future will need to be lifelong learners with the skills to engage with emerging science and translate it into clinical care. How medical schools can best prepare students for ongoing scientific change remains unclear. Adding to the challenge is reduced time allocated to basic science in curricula and rapid expansion of relevant scientific fields. A return to science with greater depth after clinical clerkships has been suggested, although few schools have adopted such curricula and implementation can present challenges. The authors describe an innovation at Harvard Medical School, the Advanced Integrated Science Courses (AISCs), which are taken after core clerkships. Students are required to take 2 such courses, which are offered in a variety of topics. Rather than factual content, the learning objectives are a set of generalizable skills to enable students to critically evaluate emerging research and its relationship to medical practice. Making these generalizable skills the defining principle of the courses has several important advantages: it allows standardization of acquired skills to be combined with diverse course topics ranging from basic to translational and population sciences; students can choose courses and projects aligned with their interests, thereby enhancing engagement, curiosity, and career relevance; schools can tailor course offerings to the interests of local faculty; and the generalizable skills delineate a unique purpose of these courses within the overall medical school curriculum. For the 3 years AISCs have been offered, students rated the courses highly and reported learning the intended skill set effectively. The AISC concept addresses the challenge of preparing students for this era of rapidly expanding science and should be readily adaptable to other medical schools.

Chromosome nondisjunction yields tetraploid rather than aneuploid cells in human cell lines.

Although mutations in cell cycle regulators or spindle proteins can perturb chromosome segregation, the causes and consequences of spontaneous mitotic chromosome nondisjunction in human cells are not well understood. It has been assumed that nondisjunction of a chromosome during mitosis will yield two aneuploid daughter cells. Here we show that chromosome nondisjunction is tightly coupled to regulation of cytokinesis in human cell lines, such that nondisjunction results in the formation of tetraploid rather than aneuploid cells. We observed that spontaneously arising binucleated cells exhibited chromosome mis-segregation rates up to 166-fold higher than the overall mitotic population. Long-term imaging experiments indicated that most binucleated cells arose through a bipolar mitosis followed by regression of the cleavage furrow hours later. Nondisjunction occurred with high frequency in cells that became binucleated by furrow regression, but not in cells that completed cytokinesis to form two mononucleated cells. Our findings indicate that nondisjunction does not directly yield aneuploid cells, but rather tetraploid cells that may subsequently become aneuploid through further division. The coupling of spontaneous segregation errors to furrow regression provides a potential explanation for the prevalence of hyperdiploid chromosome number and centrosome amplification observed in many cancers.

Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity.

Deubiquitylating enzymes (DUBs) counteract ubiquitylation to control stability or activity of substrates. Identification of DUB substrates is challenging because multiple DUBs can act on the same substrate, thwarting genetic approaches. Here, we circumvent redundancy by chemically inhibiting multiple DUBs simultaneously in Xenopus egg extract. We used quantitative mass spectrometry to identify proteins whose ubiquitylation or stability is altered by broad DUB inhibition, and confirmed their DUB-dependent regulation with human orthologs, demonstrating evolutionary conservation. We next extended this method to profile DUB specificity. By adding recombinant DUBs to extract where DUB activity was broadly inhibited, but ubiquitylation and degradation were active at physiological rates, we profiled the ability of DUBs to rescue degradation of these substrates. We found that USP7 has a unique ability to broadly antagonize degradation. Together, we present an approach to identify DUB substrates and characterize DUB specificity that overcomes challenges posed by DUB redundancy.