HAPSTR1 localizes HUWE1 to the nucleus to limit stress signaling pathways.

HUWE1 is a large, enigmatic HECT-domain ubiquitin ligase implicated in the regulation of diverse pathways, including DNA repair, apoptosis, and differentiation. How HUWE1 engages its structurally diverse substrates and how HUWE1 activity is regulated are unknown. Using unbiased quantitative proteomics, we find that HUWE1 targets substrates in a largely cell-type-specific manner. However, we identify C16orf72/HAPSTR1 as a robust HUWE1 substrate in multiple cell lines. Previously established physical and genetic interactions between HUWE1 and HAPSTR1 suggest that HAPSTR1 positively regulates HUWE1 function. Here, we show that HAPSTR1 is required for HUWE1 nuclear localization and nuclear substrate targeting. Nuclear HUWE1 is required for both cell proliferation and modulation of stress signaling pathways, including p53 and nuclear factor κB (NF-κB)-mediated signaling. Combined, our results define a role for HAPSTR1 in gating critical nuclear HUWE1 functions.

Solenoid architecture of HUWE1 contributes to ligase activity and substrate recognition.

HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival factors, including Mcl1, p53, DDIT4, and Myc. Although mutations in HUWE1 and related HECT ligases are widely implicated in human disease, our molecular understanding remains limited. Here we present a comprehensive investigation of full-length HUWE1, deepening our understanding of this class of enzymes. The N-terminal ∼3,900 amino acids of HUWE1 are indispensable for proper ligase function, and our cryo-EM structures of HUWE1 offer a complete molecular picture of this large HECT ubiquitin ligase. HUWE1 forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. Structures of HUWE1 variants linked to neurodevelopmental disorders as well as of HUWE1 bound to a model substrate link the functions of this essential enzyme to its three-dimensional organization.

Dynamic regulation of dynein localization revealed by small molecule inhibitors of ubiquitination enzymes.

Cytoplasmic dynein is a minus-end-directed microtubule-based motor that acts at diverse subcellular sites. During mitosis, dynein localizes simultaneously to the mitotic spindle, spindle poles, kinetochores and the cell cortex. However, it is unclear what controls the relative targeting of dynein to these locations. As dynein is heavily post-translationally modified, we sought to test a role for these modifications in regulating dynein localization. We find that dynein rapidly and strongly accumulates at mitotic spindle poles following treatment with NSC697923, a small molecule that inhibits the ubiquitin E2 enzyme, Ubc13, or treatment with PYR-41, a ubiquitin E1 inhibitor. Subsets of dynein regulators such as Lis1, ZW10 and Spindly accumulate at the spindle poles, whereas others do not, suggesting that NSC697923 differentially affects specific dynein populations. We additionally find that dynein relocalization induced by NSC697923 or PYR-41 can be suppressed by simultaneous treatment with the non-selective deubiquitinase inhibitor, PR-619. However, we did not observe altered dynein localization following treatment with the selective E1 inhibitor, TAK-243. Although it is possible that off-target effects of NSC697923 and PYR-41 are responsible for the observed changes in dynein localization, the rapid relocalization upon drug treatment highlights the highly dynamic nature of dynein regulation during mitosis.

The kinetochore-microtubule interface at a glance.

Accurate chromosome segregation critically depends on the formation of attachments between microtubule polymers and each sister chromatid. The kinetochore is the macromolecular complex that assembles at the centromere of each chromosome during mitosis and serves as the link between the DNA and the microtubules. In this Cell Science at a Glance article and accompanying poster, we discuss the activities and molecular players that are involved in generating kinetochore-microtubule attachments, including the initial stages of lateral kinetochore-microtubule interactions and maturation to stabilized end-on attachments. We additionally explore the features that contribute to the ability of the kinetochore to track with dynamic microtubules. Finally, we examine the contributions of microtubule-associated proteins to the organization and stabilization of the mitotic spindle and the control of microtubule dynamics.

Nde1 promotes diverse dynein functions through differential interactions and exhibits an isoform-specific proteasome association.

Nde1 is a key regulator of cytoplasmic dynein, binding directly to both dynein itself and the dynein adaptor, Lis1. Nde1 and Lis1 are thought to function together to promote dynein function, yet mutations in each result in distinct neurodevelopment phenotypes. To reconcile these phenotypic differences, we sought to dissect the contribution of Nde1 to dynein regulation and explore the cellular functions of Nde1. Here we show that an Nde1-Lis1 interaction is required for spindle pole focusing and Golgi organization but is largely dispensable for centrosome placement, despite Lis1 itself being required. Thus, diverse functions of dynein rely on distinct Nde1- and Lis1-mediated regulatory mechanisms. Additionally, we discovered a robust, isoform-specific interaction between human Nde1 and the 26S proteasome and identify precise mutations in Nde1 that disrupt the proteasome interaction. Together, our work suggests that Nde1 makes unique contributions to human neurodevelopment through its regulation of both dynein and proteasome function.

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces.

The macromolecular kinetochore functions to generate interactions between chromosomal DNA and spindle microtubules [1]. To facilitate chromosome movement and segregation, kinetochores must maintain associations with both growing and shrinking microtubule ends. It is critical to define the proteins and their properties that allow kinetochores to associate with dynamic microtubules. The kinetochore-localized human Ska1 complex binds to microtubules and tracks with depolymerizing microtubule ends [2]. We now demonstrate that the Ska1 complex also autonomously tracks with growing microtubule ends in vitro, a key property that would allow this complex to act at kinetochores to mediate persistent associations with dynamic microtubules. To define the basis for Ska1 complex interactions with dynamic microtubules, we investigated the tubulin-binding properties of the Ska1 microtubule binding domain. In addition to binding to the microtubule lattice and dolastatin-induced protofilament-like structures, we demonstrate that the Ska1 microtubule binding domain can associate with soluble tubulin heterodimers and promote assembly of oligomeric ring-like tubulin structures. We generated mutations on distinct surfaces of the Ska1 microtubule binding domain that disrupt binding to soluble tubulin but do not prevent microtubule binding. These mutants display compromised microtubule tracking activity in vitro and result in defective chromosome alignment and mitotic progression in cells using a CRISPR/Cas9-based replacement assay. Our work supports a model in which multiple surfaces of Ska1 interact with diverse tubulin substrates to associate with dynamic microtubule polymers and facilitate optimal chromosome segregation.

Astrin-SKAP complex reconstitution reveals its kinetochore interaction with microtubule-bound Ndc80.

Chromosome segregation requires robust interactions between the macromolecular kinetochore structure and dynamic microtubule polymers. A key outstanding question is how kinetochore-microtubule attachments are modulated to ensure that bi-oriented attachments are selectively stabilized and maintained. The Astrin-SKAP complex localizes preferentially to properly bi-oriented sister kinetochores, representing the final outer kinetochore component recruited prior to anaphase onset. Here, we reconstitute the 4-subunit Astrin-SKAP complex, including a novel MYCBP subunit. Our work demonstrates that the Astrin-SKAP complex contains separable kinetochore localization and microtubule binding domains. In addition, through cross-linking analysis in human cells and biochemical reconstitution, we show that the Astrin-SKAP complex binds synergistically to microtubules with the Ndc80 complex to form an integrated interface. We propose a model in which the Astrin-SKAP complex acts together with the Ndc80 complex to stabilize correctly formed kinetochore-microtubule interactions.

Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M or UBC12) and DCN1 (DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl-amide-binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress anchorage-independent growth of a cell line with DCN1 amplification. Overall, our data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets and provide insights into targeting multiprotein E2-E3 ligases.

Chromosome Segregation: A Spatial Code to Correct Kinetochore-Microtubule Attachments.

Erroneous kinetochore-microtubule interactions must be detected and corrected before a cell enters anaphase to prevent chromosome mis-segregation. Two new studies describe an Aurora A-mediated error correction mechanism based on the spatial position of a chromosome within the mitotic spindle.

Structure of a RING E3 trapped in action reveals ligation mechanism for the ubiquitin-like protein NEDD8.

Most E3 ligases use a RING domain to activate a thioester-linked E2∼ubiquitin-like protein (UBL) intermediate and promote UBL transfer to a remotely bound target protein. Nonetheless, RING E3 mechanisms matching a specific UBL and acceptor lysine remain elusive, including for RBX1, which mediates NEDD8 ligation to cullins and >10% of all ubiquitination. We report the structure of a trapped RING E3-E2∼UBL-target intermediate representing RBX1-UBC12∼NEDD8-CUL1-DCN1, which reveals the mechanism of NEDD8 ligation and how a particular UBL and acceptor lysine are matched by a multifunctional RING E3. Numerous mechanisms specify cullin neddylation while preventing noncognate ubiquitin ligation. Notably, E2-E3-target and RING-E2∼UBL modules are not optimized to function independently, but instead require integration by the UBL and target for maximal reactivity. The UBL and target regulate the catalytic machinery by positioning the RING-E2∼UBL catalytic center, licensing the acceptor lysine, and influencing E2 reactivity, thereby driving their specific coupling by a multifunctional RING E3.

Kinetochore genes are coordinately up-regulated in human tumors as part of a FoxM1-related cell division program.

The key player in directing proper chromosome segregation is the macromolecular kinetochore complex, which mediates DNA-microtubule interactions. Previous studies testing individual kinetochore genes documented examples of their overexpression in tumors relative to normal tissue, leading to proposals that up-regulation of specific kinetochore genes may promote tumor progression. However, kinetochore components do not function in isolation, and previous studies did not comprehensively compare the expression behavior of kinetochore components. Here we analyze the expression behavior of the full range of human kinetochore components in diverse published expression compendia, including normal tissues and tumor samples. Our results demonstrate that kinetochore genes are rarely overexpressed individually. Instead, we find that core kinetochore genes are coordinately regulated with other cell division genes under virtually all conditions. This expression pattern is strongly correlated with the expression of the forkhead transcription factor FoxM1, which binds to the majority of cell division promoters. These observations suggest that kinetochore gene up-regulation in cancer reflects a general activation of the cell division program and that altered expression of individual kinetochore genes is unlikely to play a causal role in tumorigenesis.

Structural conservation of distinctive N-terminal acetylation-dependent interactions across a family of mammalian NEDD8 ligation enzymes.

Little is known about molecular recognition of acetylated N termini, despite prevalence of this modification among eukaryotic cytosolic proteins. We report that the family of human DCN-like (DCNL) co-E3s, which promote ligation of the ubiquitin-like protein NEDD8 to cullin targets, recognizes acetylated N termini of the E2 enzymes UBC12 and UBE2F. Systematic biochemical and biophysical analyses reveal 40- and 10-fold variations in affinities among different DCNL-cullin and DCNL-E2 complexes, contributing to varying efficiencies of different NEDD8 ligation cascades. Structures of DCNL2 and DCNL3 complexes with N-terminally acetylated peptides from UBC12 and UBE2F illuminate a common mechanism by which DCNL proteins recognize N-terminally acetylated E2s and how selectivity for interactions dependent on N-acetyl-methionine are established through side chains recognizing distal residues. Distinct preferences of UBC12 and UBE2F peptides for inhibiting different DCNLs, including the oncogenic DCNL1 protein, suggest it may be possible to develop small molecules blocking specific N-acetyl-methionine-dependent protein interactions.

Structural basis for a reciprocal regulation between SCF and CSN.

Skp1-Cul1-Fbox (SCF) E3 ligases are activated by ligation to the ubiquitin-like protein Nedd8, which is reversed by the deneddylating Cop9 signalosome (CSN). However, CSN also promotes SCF substrate turnover through unknown mechanisms. Through biochemical and electron microscopy analyses, we determined molecular models of CSN complexes with SCF(Skp2/Cks1) and SCF(Fbw7) and found that CSN occludes both SCF functional sites-the catalytic Rbx1-Cul1 C-terminal domain and the substrate receptor. Indeed, CSN binding prevents SCF interactions with E2 enzymes and a ubiquitination substrate, and it inhibits SCF-catalyzed ubiquitin chain formation independent of deneddylation. Importantly, CSN prevents neddylation of the bound cullin, unless binding of a ubiquitination substrate triggers SCF dissociation and neddylation. Taken together, the results provide a model for how reciprocal regulation sensitizes CSN to the SCF assembly state and inhibits a catalytically competent SCF until a ubiquitination substrate drives its own degradation by displacing CSN, thereby promoting cullin neddylation and substrate ubiquitination.

N-terminal acetylation acts as an avidity enhancer within an interconnected multiprotein complex.

Although many eukaryotic proteins are amino (N)-terminally acetylated, structural mechanisms by which N-terminal acetylation mediates protein interactions are largely unknown. Here, we found that N-terminal acetylation of the E2 enzyme, Ubc12, dictates distinctive E3-dependent ligation of the ubiquitin-like protein Nedd8 to Cul1. Structural, biochemical, biophysical, and genetic analyses revealed how complete burial of Ubc12's N-acetyl-methionine in a hydrophobic pocket in the E3, Dcn1, promotes cullin neddylation. The results suggest that the N-terminal acetyl both directs Ubc12's interactions with Dcn1 and prevents repulsion of a charged N terminus. Our data provide a link between acetylation and ubiquitin-like protein conjugation and define a mechanism for N-terminal acetylation-dependent recognition.

Synthesis and structure-activity evaluation of isatin-ß-thiosemicarbazones with improved selective activity toward multidrug-resistant cells expressing P-glycoprotein.

Cancer multidrug resistance (MDR) mediated by ATP-binding cassette (ABC) transporters presents a significant unresolved clinical challenge. One strategy to resolve MDR is to develop compounds that selectively kill cells overexpressing the efflux transporter P-glycoprotein (MDR1, P-gp, ABCB1). We have previously reported structure-activity studies based around the lead compound NSC73306 (1, 1-isatin-4-(4'-methoxyphenyl)-3-thiosemicarbazone, 4.3-fold selective). Here we sought to extend this work on MDR1-selective analogues by establishing whether 1 showed "robust" activity against a range of cell lines expressing P-gp. We further aimed to synthesize and test analogues with varied substitution at the N4-position, and substitution around the N4-phenyl ring of isatin-ß-thiosemicarbazones (IBTs), to identify compounds with increased MDR1-selectivity. Compound 1 demonstrated MDR1-selectivity against all P-gp-expressing cell lines examined. This selectivity was reversed by inhibitors of P-gp ATPase activity. Structural variation at the 4'-phenyl position of 1 yielded compounds of greater MDR1-selectivity. Two of these analogues, 1-isatin-4-(4'-nitrophenyl)-3-thiosemicarbazone (22, 8.3-fold selective) and 1-isatin-4-(4'-tert-butyl phenyl)-3-thiosemicarbazone (32, 14.8-fold selective), were selected for further testing and were found to retain the activity profile of 1. These compounds are the most active IBTs identified to date.

Serotonin transporter gene (5-HTTLPR) polymorphisms are associated with emotional modulation of pain but not emotional modulation of spinal nociception.

The short allele of the serotonin transporter gene (5-HTTLPR) is associated with greater negative emotionality. Given that emotion modulates pain, short allele carriers (s-carriers) may also demonstrate altered pain modulation. The present study used a well-validated emotional picture-viewing paradigm to modulate pain and the nociceptive flexion reflex (NFR, a measure of spinal nociception) in 144 healthy genotyped participants. As expected, pain/NFR responses were largest during unpleasant pictures and smallest during pleasant pictures. However, relative to l/l-carriers, s-carriers demonstrated greater pain inhibition during pleasant pictures and greater pain facilitation during unpleasant pictures. Neither emotional modulation of NFR nor NFR threshold was associated with 5-HTTLPR polymorphisms. Results also indicated that men who were s-carriers had a higher pain threshold and tolerance than other participants. Taken together, our results indicate 5-HTTLPR polymorphisms may influence pain modulation at the supraspinal (not spinal) level; however, the influence on pain sensitivity may be sex-specific.

A dual E3 mechanism for Rub1 ligation to Cdc53.

In ubiquitin-like protein (UBL) cascades, a thioester-linked E2∼UBL complex typically interacts with an E3 enzyme for UBL transfer to the target. Here we demonstrate a variant mechanism, whereby the E2 Ubc12 functions with two E3s, Hrt1 and Dcn1, for ligation of the UBL Rub1 to Cdc53's WHB subdomain. Hrt1 functions like a conventional RING E3, with its N terminus recruiting Cdc53 and C-terminal RING activating Ubc12∼Rub1. Dcn1's "potentiating neddylation" domain (Dcn1(P)) acts as an additional E3, reducing nonspecific Hrt1-mediated Ubc12∼Rub1 discharge and directing Ubc12's active site to Cdc53. Crystal structures of Dcn1(P)-Cdc53(WHB) and Ubc12 allow modeling of a catalytic complex, supported by mutational data. We propose that Dcn1's interactions with both Cdc53 and Ubc12 would restrict the otherwise flexible Hrt1 RING-bound Ubc12∼Rub1 to a catalytically competent orientation. Our data reveal mechanisms by which two E3s function synergistically to promote UBL transfer from one E2 to a target.

Blood lactate and hormonal responses to prototype flywheel ergometer workouts.

The purpose of the study was to compare blood lactate and hormonal responses with flywheel ergometer (FERG) leg presses for preliminary assessment of workouts best suited for future in-flight resistance exercise. Comprised of 10 repetition sets, the workouts entailed 3 sets of concentric and eccentric (CE3) actions, or concentric-only actions done for 3 (CO3) or 6 (CO6) sets. Methods employed included assessment of blood lactate concentrations ([BLa-]) before and 5 minutes postexercise. Venous blood was also collected before and at 1 and 30 minutes postexercise to assess growth hormone, testosterone, cortisol concentrations ([GH], [T], [C]) and [T/C] ratios. [BLa-] were compared with 2 (time) x 3 (workout) analysis of variance. Hormones were assessed with 2 (gender) x 3 (time) x 3 (workout) analysis of covariances. Results showed [BLa-] had a time effect. Growth hormone concentration showed gender x workout, gender x time, and workout x time interactions, whereas [T] had a 3-way interaction. [C] had gender, time, and workout effects. [T/C] yielded a gender x time interaction. It was concluded that, because CO6 and CE3 yielded similar anabolic hormonal data but the latter had a lower [C] 30 minutes postexercise, CE3 served as the best workout. Although the FERG was originally designed for microgravity, the effort put forth by current subjects was like that for workouts aimed at greater athletic performance and conditioning. Practical applications suggest that eccentric actions should be used for FERG workouts geared toward muscle mass and strength improvement.

Impact of acceleration on blood lactate values derived from high-speed resistance exercise.

Acceleration, or an increase in the rate of movement, is integral to success in many sports. Improvements in acceleration often entail workouts done at intensities that elicit higher blood lactate concentrations (BLa). The purpose of the study is to assess the impact of acceleration on BLa. Methods required subjects (n = 45) to perform 4 workouts that each involved two 1-minute sets of hip- and knee-extension repetitions on an inertial exercise trainer (Impulse Training Systems, Newnan, Georgia). Subjects performed 2 workouts comprised solely of phasic or tonic repetitions; their sequence was randomized to prevent an order effect. Before and 5 minutes after exercise, subjects' BLa were assessed with a calibrated analyzer (Sports Resource Group, Hawthorne, New York). Post and delta (post-pre) BLa both served as criterion measures for multivariate analysis. Average and peak acceleration values, derived from both phasic and tonic workouts, served as predictor variables. Results showed statistical significance (p < 0.05; R = 0.2534) and yielded the following prediction equation from phasic workouts: delta BLa = 1.40 + 1.116 (average acceleration set 1)--0.011 (peak acceleration set 1)--0.634 (average acceleration set 2) + 0.005 (peak acceleration set 2). Conclusions suggest delta BLa variance, which represents the increase of the metabolite incurred from workouts, is most easily explained by average acceleration values, which describes the mean increase in the rate of movement from phasic workouts. To improve an athlete's tolerance for acceleration-induced BLa increases, workouts should be tailored with respect to the muscles involved and the duration of exercise bouts of their chosen sport.

Growth hormone levels after flywheel ergometer exercise: correlation with independent variables.

INTRODUCTION: In-flight muscle mass and strength losses are likely exacerbated by low growth hormone (GH) concentrations. Factors associated with exercise may foretell resultant GH levels and thereby help blunt future mass and strength losses. METHODS: To assess the ability of variables to predict GH variance from resistive exercise done on a flywheel ergometer (FE) designed for in-flight exercise, subjects (N=17) performed three types of workouts on the device. With a randomized design, subjects performed the workouts with the intent to determine if changes in post-exercise GH concentrations are impacted by contractile mode and workload. Body mass, blood lactate (BLa-) concentrations, and peak angular velocity (PAV), average power (AP), and total work (TW) from workouts attempted to predict GH variance. Pre-exercise blood draws, and at 1 and 30 min after workouts, were used to determine GH concentrations. BLa- levels were measured before workouts and at 5 min post-exercise. Delta (8, post-pre) and 30-min post-workout GH levels served as criterion variables. RESULTS: Multivariate regression with an alpha < or = 0.05 yielded the following significant prediction equation: deltaGH = 13.64 - 0.014 (body mass) - 0.607 (post-exercise BLa-) + 0.659 (deltaBLa-) - 0.624(PAV) + 0.653(TW) + 0.147(AP). DISCUSSION: Univariate correlations show body mass, deltaBLa-, and TW were the best predictors of deltaGH variance. Future research should also attempt to identify additional variables that account for the unexplained GH variance from FE workouts.