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.

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.

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.

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.

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.

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.

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.

The Harvard Medical School Pathways Curriculum: Reimagining Developmentally Appropriate Medical Education for Contemporary Learners.

As the U.S. health care system changes and technology alters how doctors work and learn, medical schools and their faculty are compelled to modify their curricula and teaching methods. In this article, educational leaders and key faculty describe how the Pathways curriculum was conceived, designed, and implemented at Harvard Medical School. Faculty were committed to the principle that educators should focus on how students learn and their ability to apply what they learn in the evaluation and care of patients. Using the best evidence from the cognitive sciences about adult learning, they made major changes in the pedagogical approach employed in the classroom and clinic. The curriculum was built upon 4 foundational principles: to enhance critical thinking and provide developmentally appropriate content; to ensure both horizontal integration between courses and vertical integration between phases of the curriculum; to engage learners, foster curiosity, and reinforce the importance of student ownership and responsibility for their learning; and to support students' transformation to a professional dedicated to the care of their patients and to their obligations for lifelong, self-directed learning.The practice of medicine is rapidly evolving and will undoubtedly change in multiple ways over the career of a physician. By emphasizing personal responsibility, professionalism, and thinking skills over content transfer, the authors believe this curriculum will prepare students not only for the first day of practice but also for an uncertain future in the biological sciences, health and disease, and the nation's health care system, which they will encounter in the decades to come.

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.

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.

Inhibiting the anaphase promoting complex/cyclosome induces a metaphase arrest and cell death in multiple myeloma cells.

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.

Defective sister chromatid cohesion is synthetically lethal with impaired APC/C function.

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 p31(comet). 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.

Substrate degradation by the proteasome: a single-molecule kinetic analysis.

To address how the configuration of conjugated ubiquitins determines the recognition of substrates by the proteasome, we analyzed the degradation kinetics of substrates with chemically defined ubiquitin configurations. Contrary to the view that a tetraubiquitin chain is the minimal signal for efficient degradation, we find that distributing the ubiquitins as diubiquitin chains provides a more efficient signal. To understand how the proteasome actually discriminates among ubiquitin configurations, we developed single-molecule assays that distinguished intermediate steps of degradation kinetically. The level of ubiquitin on a substrate drives proteasome-substrate interaction, whereas the chain structure of ubiquitin affects translocation into the axial channel on the proteasome. Together these two features largely determine the susceptibility of substrates for proteasomal degradation.

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.

The G2/M regulator histone demethylase PHF8 is targeted for degradation by the anaphase-promoting complex containing CDC20.

Monomethylated histone H4 lysine 20 (H4K20me1) is tightly regulated during the cell cycle. The H4K20me1 demethylase PHF8 transcriptionally regulates many cell cycle genes and is therefore predicted to play key roles in the cell cycle. Here, we show that PHF8 protein levels are the highest during G2 phase and mitosis, and we found PHF8 protein stability to be regulated by the ubiquitin-proteasome system. Purification of the PHF8 complex led to the identification of many subunits of the anaphase-promoting complex (APC) associated with PHF8. We showed that PHF8 interacts with the CDC20-containing APC (APC(cdc20)) primarily during mitosis. In addition, we defined a novel, KEN- and D-box-independent, LXPKXLF motif on PHF8 that is required for binding to CDC20. Through various in vivo and in vitro assays, we demonstrate that mutations of the LXPKXLF motif abrogate polyubiquitylation of PHF8 by the APC. APC substrates are typically cell cycle regulators, and consistent with this, the loss of PHF8 leads to prolonged G2 phase and defective mitosis. Furthermore, we provide evidence that PHF8 plays an important role in transcriptional activation of key G2/M genes during G2 phase. Taken together, these findings suggest that PHF8 is regulated by APC(cdc20) and plays an important role in the G2/M transition.

Determinants of human cyclin B1 association with mitotic chromosomes.

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.

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.

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response.

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.