Crystal Structure of a Ube2S-Ubiquitin Conjugate

Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a “donor” ubiquitin and a primary amino group of an “acceptor” ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface

A brief description of geological and geophysical exploration of the Marysville geothermal area

Extensive geological and geophysical surveys were carried out at the Marysville geothermal area during 1973 and 1974. The area has high heat flow (up to microcalories per square centimeter-second, a negative gravity anomaly, high electrical resistivity, low seismic ground noise, and nearby microseismic activity. Significant magnetic and infrared anomalies are not associated with the geothermal area. The geothermal anomaly occupies the axial portion of a dome in Precambrian sedimentary rocks intruded by Cretaceous and Cenozoic granitic rocks. The results from a 2.4-km-deep test well indicate that the cause of the geothermal anomaly is hydrothermal convection in a Cenozoic intrusive. A maximum temperature of 95 C was measured at a depth of 500 m in the test well

Violent and victimized bodies: sexual violence policy in England and Wales

This paper uses the notion of the body to frame an archaeology of sexual violence policy in England and Wales, applying and developing Pillow’s ideas. It argues that the dominant construction is of sexual violence as an individualized crime, with the solution being for a survivor to report, and with support often instrumentalized in relation to criminal justice objectives. However, criminal justice proceedings can intensify or create further trauma for sexual violence survivors. Furthermore, in addition to criminalizing the violent body and supporting the victimized one, there is a need for policy to produce alternative types of bodies through preventative interventions. Much sexual violence is situated within (hetero) sexual dynamics constructing a masculine aggressor and a feminine body which eventually yields. Prevention must therefore focus on developing embodied boundaries, and narratives at the margins of policy could underpin such efforts

The role of CDC48 in the retro-translocation of non-ubiquitinated toxin substrates in plant cells

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterise the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that while RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesised RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated

A survey of practices for the use of electronic health records to support research recruitment

Electronic health records (EHRs) provide great promise for identifying cohorts and enhancing research recruitment. Such approaches are sorely needed, but there are few descriptions in the literature of prevailing practices to guide their use. A multidisciplinary workgroup was formed to examine current practices in the use of EHRs in recruitment and to propose future directions. The group surveyed consortium members regarding current practices. Over 98% of the Clinical and Translational Science Award Consortium responded to the survey. Brokered and self-service data warehouse access are in early or full operation at 94% and 92% of institutions, respectively, whereas, EHR alerts to providers and to research teams are at 45% and 48%, respectively, and use of patient portals for research is at 20%. However, these percentages increase significantly to 88% and above if planning and exploratory work were considered cumulatively. For most approaches, implementation reflected perceived demand. Regulatory and workflow processes were similarly varied, and many respondents described substantive restrictions arising from logistical constraints and limitations on collaboration and data sharing. Survey results reflect wide variation in implementation and approach, and point to strong need for comparative research and development of best practices to protect patients and facilitate interinstitutional collaboration and multisite research

Expression of Constitutively Active CDK1 Stabilizes APC-Cdh1 Substrates and Potentiates Premature Spindle Assembly and Checkpoint Function in G1 Cells

Mitotic progression in eukaryotic cells depends upon the activation of cyclin-dependent kinase 1 (CDK1), followed by its inactivation through the anaphase-promoting complex (APC)/cyclosome-mediated degradation of M-phase cyclins. Previous work revealed that expression of a constitutively active CDK1 (CDK1AF) in HeLa cells permitted their division, but yielded G1 daughter cells that underwent premature S-phase and early mitotic events. While CDK1AF was found to impede the sustained activity of APC-Cdh1, it was unknown if this defect improperly stabilized mitotic substrates and contributed to the occurrence of these premature M phases. Here, we show that CDK1AF expression in HeLa cells improperly stabilized APC-Cdh1 substrates in G1-phase daughter cells, including mitotic kinases and the APC adaptor, Cdc20. Division of CDK1AF-expressing cells produced G1 daughters with an accelerated S-phase onset, interrupted by the formation of premature bipolar spindles capable of spindle assembly checkpoint function. Further characterization of these phenotypes induced by CDK1AF expression revealed that this early spindle formation depended upon premature CDK1 and Aurora B activities, and their inhibition induced rapid spindle disassembly. Following its normal M-phase degradation, we found that the absence of Wee1 in these prematurely cycling daughter cells permitted the endogenous CDK1 to contribute to these premature mitotic events, since expression of a non-degradable Wee1 reduced the number of cells that exhibited premature cyclin B1oscillations. Lastly, we discovered that Cdh1-ablated cells could not be forced into a premature M phase, despite cyclin B1 overexpression and proteasome inhibition. Together, these results demonstrate that expression of constitutively active CDK1AF hampers the destruction of critical APC-Cdh1 targets, and that this type of condition could prevent newly divided cells from properly maintaining a prolonged interphase state. We propose that this more subtle type of defect in activity of the APC-driven negative-feedback loop may have implications for triggering genome instability and tumorigenesis

A Putative Homologue of CDC20/CDH1 in the Malaria Parasite Is Essential for Male Gamete Development

Cell-cycle progression is governed by a series of essential regulatory proteins. Two major regulators are cell-division cycle protein 20 (CDC20) and its homologue, CDC20 homologue 1 (CDH1), which activate the anaphase-promoting complex/cyclosome (APC/C) in mitosis, and facilitate degradation of mitotic APC/C substrates. The malaria parasite, Plasmodium, is a haploid organism which, during its life-cycle undergoes two stages of mitosis; one associated with asexual multiplication and the other with male gametogenesis. Cell-cycle regulation and DNA replication in Plasmodium was recently shown to be dependent on the activity of a number of protein kinases. However, the function of cell division cycle proteins that are also involved in this process, such as CDC20 and CDH1 is totally unknown. Here we examine the role of a putative CDC20/CDH1 in the rodent malaria Plasmodium berghei (Pb) using reverse genetics. Phylogenetic analysis identified a single putative Plasmodium CDC20/CDH1 homologue (termed CDC20 for simplicity) suggesting that Plasmodium APC/C has only one regulator. In our genetic approach to delete the endogenous cdc20 gene of P. berghei, we demonstrate that PbCDC20 plays a vital role in male gametogenesis, but is not essential for mitosis in the asexual blood stage. Furthermore, qRT-PCR analysis in parasite lines with deletions of two kinase genes involved in male sexual development (map2 and cdpk4), showed a significant increase in cdc20 transcription in activated gametocytes. DNA replication and ultra structural analyses of cdc20 and map2 mutants showed similar blockage of nuclear division at the nuclear spindle/kinetochore stage. CDC20 was phosphorylated in asexual and sexual stages, but the level of modification was higher in activated gametocytes and ookinetes. Changes in global protein phosphorylation patterns in the Δcdc20 mutant parasites were largely different from those observed in the Δmap2 mutant. This suggests that CDC20 and MAP2 are both likely to play independent but vital roles in male gametogenesis

Overexpression of UbcH10 alternates the cell cycle profile and accelerate the tumor proliferation in colon cancer

<p>Abstract</p> <p>Background</p> <p>UbcH10 participates in proper metaphase to anaphase transition, and abrogation of UbcH10 results in the premature separation of sister chromatids. To assess the potential role of UbcH10 in colon cancer progression, we analyzed the clinicopathological relevance of UbcH10 in colon cancer.</p> <p>Methods</p> <p>We firstly screened the expression profile of UbcH10 in various types of cancer tissues as well as cell lines. Thereafter, using the colon cancer cells line, we manipulated the expression of UbcH10 and evaluated the cell cycle profile and cellular proliferations. Furthermore, the clinicopathological significance of UbcH10 was immunohistologically evaluated in patients with colon cancer. Statistical analysis was performed using the student's t-test and Chi-square test.</p> <p>Results</p> <p>Using the colon cancer cells, depletion of UbcH10 resulted in suppression of cellular growth whereas overexpression of UbcH10 promoted the cellular growth and oncogenic cellular growth. Mitotic population was markedly alternated by the manipulation of UbcH10 expression. Immunohistochemical analysis indicated that UbcH10 was significantly higher in colon cancer tissue compared with normal colon epithelia. Furthermore, the clinicopathological evaluation revealed that UbcH10 was associated with high-grade histological tumors.</p> <p>Conclusion</p> <p>The results show the clinicopathological significance of UbcH10 in the progression of colon cancer. Thus UbcH10 may act as a novel biomarker in patients with colon cancer.</p

Increased sinusoidal flow is not the primary stimulus to liver regeneration

Background: Hemodynamic changes in the liver remnant following partial hepatectomy (PHx) have been suggested to be a primary stimulus in triggering liver regeneration. We hypothesized that it is the increased sinusoidal flow per se and hence the shear-stress stimulus on the endothelial surface within the liver remnant which is the main stimulus to regeneration. In order to test this hypothesis we wanted to increase the sinusoidal flow without performing a concomitant liver resection. Accordingly, we constructed an aorto-portal shunt to the left portal vein branch creating a standardized four-fold increase in flow to segments II, III and IV. The impact of this manipulation was studied in both an acute model (6 animals, 9 hours) using a global porcine cDNA microarray chip and in a chronic model observing weight and histological changes (7 animals, 3 weeks). Results: Gene expression profiling from the shunted segments does not suggest that increased sinusoidal flow per se results in activation of genes promoting mitosis. Hyperperfusion over three weeks results in the whole liver gaining a supranormal weight of 3.9% of the total body weight (versus the normal 2.5%). Contrary to our hypothesis, the weight gain was observed on the non-shunted side without an increase in sinusoidal flow. Conclusions: An isolated increase in sinusoidal flow does not have the same genetic, microscopic or macroscopic impact on the liver as that seen in the liver remnant after partial hepatectomy, indicating that increased sinusoidal flow may not be a sufficient stimulus in itself for the initiation of liver regeneration

Direct Ubiquitin Independent Recognition and Degradation of a Folded Protein by the Eukaryotic Proteasomes-Origin of Intrinsic Degradation Signals

Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a) origin and identification of an intrinsic degradation signal in the substrate, b) identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c) identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably catalyzed by the ATPases. Apomyoglobin emerges as a new model substrate to further explore the role of ATPases and protein structure in proteasomal degradatio