Functional Diversity and Structural Disorder in the Human Ubiquitination Pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions. © 2013 Bhowmick et al

Flash Glucose-Sensing Technology as a Replacement for Blood Glucose Monitoring for the Management of Insulin-Treated Type 2 Diabetes: a Multicenter, Open-Label Randomized Controlled Trial

Introduction Glycemic control in participants with insulin-treated diabetes remains challenging. We assessed safety and efficacy of new flash glucose-sensing technology to replace self-monitoring of blood glucose (SMBG). Methods This open-label randomized controlled study (ClinicalTrials.gov, NCT02082184) enrolled adults with type 2 diabetes on intensive insulin therapy from 26 European diabetes centers. Following 2 weeks of blinded sensor wear, 2:1 (intervention/control) randomization (centrally, using biased-coin minimization dependant on study center and insulin administration) was to control (SMBG) or intervention (glucose-sensing technology). Participants and investigators were not masked to group allocation. Primary outcome was difference in HbA1c at 6 months in the full analysis set. Prespecified secondary outcomes included time in hypoglycemia, effect of age, and patient satisfaction. Results Participants (n = 224) were randomized (149 intervention, 75 controls). At 6 months, there was no difference in the change in HbA1c between intervention and controls: −3.1 ± 0.75 mmol/mol, [−0.29 ± 0.07% (mean ± SE)] and −3.4 ± 1.04 mmol/mol (−0.31 ± 0.09%) respectively; p = 0.8222. A difference was detected in participants aged <65 years [−5.7 ± 0.96 mmol/mol (−0.53 ± 0.09%) and −2.2 ± 1.31 mmol/mol (−0.20 ± 0.12%), respectively; p = 0.0301]. Time in hypoglycemia <3.9 mmol/L (70 mg/dL) reduced by 0.47 ± 0.13 h/day [mean ± SE (p = 0.0006)], and <3.1 mmol/L (55 mg/dL) reduced by 0.22 ± 0.07 h/day (p = 0.0014) for intervention participants compared with controls; reductions of 43% and 53%, respectively. SMBG frequency, similar at baseline, decreased in intervention participants from 3.8 ± 1.4 tests/day (mean ± SD) to 0.3 ± 0.7, remaining unchanged in controls. Treatment satisfaction was higher in intervention compared with controls (DTSQ 13.1 ± 0.50 (mean ± SE) and 9.0 ± 0.72, respectively; p < 0.0001). No serious adverse events or severe hypoglycemic events were reported related to sensor data use. Forty-two serious events [16 (10.7%) intervention participants, 12 (16.0%) controls] were not device-related. Six intervention participants reported nine adverse events for sensor-wear reactions (two severe, six moderate, one mild). Conclusion Flash glucose-sensing technology use in type 2 diabetes with intensive insulin therapy results in no difference in HbA1c change and reduced hypoglycemia, thus offering a safe, effective replacement for SMBG

Use of Flash Glucose-Sensing Technology for 12 months as a Replacement for Blood Glucose Monitoring in Insulin-treated Type 2 Diabetes

Introduction: Published evaluations of sensor glucose monitoring use in insulin treated type 2 diabetes are limited. The aim of this study was to assess the impact of flash glucose-sensing technology as a replacement for self-monitoring of blood glucose (SMBG) over a 12-month period in participants with type 2 diabetes who were on intensive insulin therapy. Methods: An open-label, randomized, controlled study in adults with type 2 diabetes on intensive insulin therapy from 26 European diabetes centers aimed at assessing flash glucose sensing technology was conducted. Participants (N = 224) were randomized (1:2 respectively) to a control group (n = 75) that used SMBG (FreeStyle Lite™) or to an intervention group (n = 149) which used sensor glucose data (FreeStyle Libre™ Flash Glucose Monitoring System) for self-management over 6 months. All intervention group participants who completed the 6-month treatment phase continued into an additional 6-month open-access phase. Results: A total of 139 intervention participants completed the 6-month treatment phase and continued into the open-access phase. At 12 months (end of open-access period), time in hypoglycemia [sensor glucose <3.9 mmol/L (70 mg/dL)] was reduced by 50% compared to baseline [−0.70 ± 1.85/24 h (mean ± standard deviation); p = 0.0002]. Nocturnal hypoglycemia [2300 to 0600 hours, <3.9 mmol/L (70 mg/dL)] was reduced by 52%; p = 0.0002. There was no change in time in range [sensor glucose 3.9–10.0 mmol/L (70–180 mg/dL)]. SMBG testing fell from a mean of 3.9 (median 3.9) times/day at baseline to 0.2 (0.0), with an average frequency of sensor scanning of 7.1 (5.7) times/day at 12 months, and mean sensor utilization was 83.6 ± 13.8% (median 88.3%) during the open-access phase. During this 6-month extension period no device-related serious adverse events were reported. Nine participants reported 16 instances of device-related adverse events (e.g. infection, allergy) and 28 participants (20.1%) experienced 134 occurrences of anticipated skin symptoms/sensor-insertion events expected with device use (e.g. erythema, itching and rash). Conclusion: The use of flash glucose-sensing technology for glycemic management in individuals with type 2 diabetes treated by intensive insulin therapy over 12 months was associated with a sustained reduction in hypoglycemia and safely and effectively replaced SMBG. Trial Registration: ClinicalTrials.gov identifier, NCT02082184

Unravelling the controls on the molybdenum isotope ratios of river waters

The molybdenum (Mo) isotope ratios (δ98/95Mo) of river waters control the δ98/95Mo values of seawater and impact on the use of Mo isotope ratios as a proxy of past redox conditions. The δ98/95Mo values of river waters vary by more than 2 ‰, yet the relative roles of lithology versus fractionation during weathering remain contested. Here, we combine measurements from river waters (δ98/95Modiss), river bed materials (δ98/95MoBM) and soils from locations with contrasting lithology. The δ98/95Mo values of river bed materials (δ98/95MoBM), set by rock type, vary by ~1 ‰ between rivers in New Zealand, the Mackenzie Basin, and Iceland. However, the difference between dissolved and solid phase Mo isotopes (Δ98/95Modiss-BM) varies from +0.3 ‰ to +1.0 ‰. We estimate Mo removal from solution using the mobile trace element rhenium and find that it correlates with Δ98/95Modiss-BM across the sample set. The adsorption of Mo to Fe-Mn-(oxyhydr) oxides can explain the observed fractionation. Together, the amount of Mo released through dissolution and taken up by (oxyhydr)oxide formation on land may cause changes in the δ98/95Mo values of rivers, driving long term changes in the Mo isotope ratios of seawater

Three-dimensional structure of a viral genome-delivery portal vertex.

DNA viruses such as bacteriophages and herpesviruses deliver their genome into and out of the capsid through large proteinaceous assemblies, known as portal proteins. Here, we report two snapshots of the dodecameric portal protein of bacteriophage P22. The 3.25-Å-resolution structure of the portal-protein core bound to 12 copies of gene product 4 (gp4) reveals a ~1.1-MDa assembly formed by 24 proteins. Unexpectedly, a lower-resolution structure of the full-length portal protein unveils the unique topology of the C-terminal domain, which forms a ~200-Å-long α-helical barrel. This domain inserts deeply into the virion and is highly conserved in the Podoviridae family. We propose that the barrel domain facilitates genome spooling onto the interior surface of the capsid during genome packaging and, in analogy to a rifle barrel, increases the accuracy of genome ejection into the host cell

Examining the impact of 11 long-standing health conditions on health-related quality of life using the EQ-5D in a general population sample

Objectives Health-related quality of life (HRQoL) measures have been increasingly used in economic evaluations for policy guidance. We investigate the impact of 11 self-reported long-standing health conditions on HRQoL using the EQ-5D in a UK sample. Methods We used data from 13,955 patients in the South Yorkshire Cohort study collected between 2010 and 2012 containing the EQ-5D, a preference-based measure. Ordinary least squares (OLS), Tobit and two-part regression analyses were undertaken to estimate the impact of 11 long-standing health conditions on HRQoL at the individual level. Results The results varied significantly with the regression models employed. In the OLS and Tobit models, pain had the largest negative impact on HRQoL, followed by depression, osteoarthritis and anxiety/nerves, after controlling for all other conditions and sociodemographic characteristics. The magnitude of coefficients was higher in the Tobit model than in the OLS model. In the two-part model, these four long-standing health conditions were statistically significant, but the magnitude of coefficients decreased significantly compared to that in the OLS and Tobit models and was ranked from pain followed by depression, anxiety/nerves and osteoarthritis. Conclusions Pain, depression, osteoarthritis and anxiety/nerves are associated with the greatest losses of HRQoL in the UK population. The estimates presented in this article should be used to inform economic evaluations when assessing health care interventions, though improvements can be made in terms of diagnostic information and obtaining longitudinal data

Targeting the hypoxic fraction of tumours using hypoxia activated prodrugs

The presence of a microenvironment within most tumours containing regions of low oxygen tension or hypoxia has profound biological and therapeutic implications. Tumour hypoxia is known to promote the development of an aggressive phenotype, resistance to both chemotherapy and radiotherapy and is strongly associated with poor clinical outcome. Paradoxically, it is recognised as a high priority target and one therapeutic strategies designed to eradicate hypoxic cells in tumours are a group of compounds known collectively as hypoxia activated prodrugs (HAPs) or bioreductive drugs. These drugs are inactive prodrugs that require enzymatic activation (typically by 1 or 2 electron oxidoreductases) to generate cytotoxic species with selectivity for hypoxic cells being determined by (i) the ability of oxygen to either reverse or inhibit the activation process and (ii) the presence of elevated expression of oxidoreductases in tumours. The concepts underpinning HAP development were established over 40 years ago and have been refined over the years to produce a new generation of HAPs that are under preclinical and clinical development. The purpose of this article is to describe current progress in the development of HAPs focusing on the mechanisms of action, preclinical properties and clinical progress of leading examples

The Pathology of EMT in Mouse Mammary Tumorigenesis

Epithelial-mesenchymal-transition (EMT) tumorigenesis in the mouse was first described over 100 years ago using various terms such as carcinosarcoma and without any comprehension of the underlying mechanisms. Such tumors have been considered artifacts of transplantation and of tissue culture. Recently, EMT tumors have been recognized in mammary glands of genetically engineered mice. This review provides a historical perspective leading to the current status in the context of some of the key molecular biology. The biology of mouse mammary EMT tumorigenesis is discussed with comparisons to human breast cancer

The tuberculosis necrotizing toxin kills macrophages by hydrolyzing NAD.

Mycobacterium tuberculosis (Mtb) induces necrosis of infected cells to evade immune responses. Recently, we found that Mtb uses the protein CpnT to kill human macrophages by secreting its C-terminal domain, named tuberculosis necrotizing toxin (TNT), which induces necrosis by an unknown mechanism. Here we show that TNT gains access to the cytosol of Mtb-infected macrophages, where it hydrolyzes the essential coenzyme NAD(+). Expression or injection of a noncatalytic TNT mutant showed no cytotoxicity in macrophages or in zebrafish zygotes, respectively, thus demonstrating that the NAD(+) glycohydrolase activity is required for TNT-induced cell death. To prevent self-poisoning, Mtb produces an immunity factor for TNT (IFT) that binds TNT and inhibits its activity. The crystal structure of the TNT-IFT complex revealed a new NAD(+) glycohydrolase fold of TNT, the founding member of a toxin family widespread in pathogenic microorganisms

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund