Regulation of peptide import through phosphorylation of Ubr1, the ubiquitin ligase of the N-end rule pathway

Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. These residues are recognized by E3 ubiquitin ligases called N-recognins. Ubr1 is the N-recognin of the yeast Saccharomyces cerevisiae. Extracellular amino acids or short peptides up-regulate the peptide transporter gene PTR2, thereby increasing the capacity of a cell to import peptides. Cup9 is a transcriptional repressor that down-regulates PTR2. The induction of PTR2 by peptides or amino acids involves accelerated degradation of Cup9 by the N-end rule pathway. We report here that the Ubr1 N-recognin, which conditionally targets Cup9 for degradation, is phosphorylated in vivo at multiple sites, including Ser300 and Tyr277. We also show that the type-I casein kinases Yck1 and Yck2 phosphorylate Ubr1 on Ser300, and thereby make possible (“prime”) the subsequent (presumably sequential) phosphorylations of Ubr1 on Ser296, Ser292, Thr288, and Tyr277 by Mck1, a kinase of the glycogen synthase kinase 3 (Gsk3) family. Phosphorylation of Ubr1 on Tyr277 by Mck1 is a previously undescribed example of a cascade-based tyrosine phosphorylation by a Gsk3-type kinase outside of autophosphorylation. We show that the Yck1/Yck2-mediated phosphorylation of Ubr1 on Ser300 plays a major role in the control of peptide import by the N-end rule pathway. In contrast to phosphorylation on Ser300, the subsequent (primed) phosphorylations, including the one on Tyr277, have at most minor effects on the known properties of Ubr1, including regulation of peptide import. Thus, a biological role of the rest of Ubr1 phosphorylation cascade remains to be identified

Gravastar energy conditions revisited

We consider the gravastar model where the vacuum phase transition between the de Sitter interior and the Schwarzschild or Schwarzschild-de Sitter exterior geometries takes place at a single spherical delta-shell. We derive sharp analytic bounds on the surface compactness (2m/r) that follow from the requirement that the dominant energy condition (DEC) holds at the shell. In the case of Schwarzschild exterior, the highest surface compactness is achieved with the stiff shell in the limit of vanishing (dark) energy density in the interior. In the case of Schwarzschild-de Sitter exterior, in addition to the gravastar configurations with the shell under surface pressure, gravastar configurations with vanishing shell pressure (dust shells), as well as configurations with the shell under surface tension, are allowed by the DEC. Respective bounds on the surface compactness are derived for all cases. We also consider the speed of sound on the shell as derived from the requirement that the shell is stable against the radial perturbations. The causality requirement (sound speed not exceeding that of light) further restricts the space of allowed gravastar configurations.Comment: LaTeX/IOP-style, 16 pages, 2 figures, changes wrt v1: motivation for eq. (6) clarified, several referecnes added (to appear in Class. Quantum Grav.

The Schro¨\ddot{o}dinger-Poisson equations as the large-N limit of the Newtonian N-body system: applications to the large scale dark matter dynamics

In this paper it is argued how the dynamics of the classical Newtonian N-body system can be described in terms of the Schr$\ddot{o}$dinger-Poisson equations in the large $N$ limit. This result is based on the stochastic quantization introduced by Nelson, and on the Calogero conjecture. According to the Calogero conjecture, the emerging effective Planck constant is computed in terms of the parameters of the N-body system as $\hbar \sim M^{5/3} G^{1/2} (N/)^{1/6}$, where is $G$ the gravitational constant, $N$ and $M$ are the number and the mass of the bodies, and $$ is their average density. The relevance of this result in the context of large scale structure formation is discussed. In particular, this finding gives a further argument in support of the validity of the Schr$\ddot{o}$dinger method as numerical double of the N-body simulations of dark matter dynamics at large cosmological scales.Comment: Accepted for publication in the Euro. Phys. J.

Nanowell-Based Immunoassays for Measuring Single-Cell Secretion: Characterization of Transport and Surface Binding

Arrays of subnanoliter wells (nanowells) provide a useful system to isolate single cells and analyze their secreted proteins. Two general approaches have emerged: one that uses open arrays and local capture of secreted proteins, and a second (called microengraving) that relies on closed arrays to capture secreted proteins on a solid substrate, which is subsequently removed from the array. However, the design and operating parameters for efficient capture from these two approaches to analyze single-cell secretion have not been extensively considered. Using numerical simulations, we analyzed the operational envelope for both open and closed formats, as a function of the spatial distribution of capture ligands, their affinities for the protein, and the rates of single-cell secretion. Based on these analyses, we present a modified approach to capture secreted proteins in-well for highly active secreting cells. This simple method for in-well detection should facilitate rapid identification of cell lines with high specific productivities.National Institutes of Health (U.S.)/National Institute of Allergy and Infectious Diseases (U.S.) (5P01AI045757)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051

A data set from flash X-ray imaging of carboxysomes

Citation: Hantke, M. F., Hasse, D., Ekeberg, T., John, K., Svenda, M., Loh, D., . . . Maia, F. R. N. C. (2016). A data set from flash X-ray imaging of carboxysomes. Scientific Data, 3. doi:10.1038/sdata.2016.61Ultra-intense femtosecond X-ray pulses from X-ray lasers permit structural studies on single particles and biomolecules without crystals. We present a large data set on inherently heterogeneous, polyhedral carboxysome particles. Carboxysomes are cell organelles that vary in size and facilitate up to 40% of Earth's carbon fixation by cyanobacteria and certain proteobacteria. Variation in size hinders crystallization. Carboxysomes appear icosahedral in the electron microscope. A protein shell encapsulates a large number of Rubisco molecules in paracrystalline arrays inside the organelle. We used carboxysomes with a mean diameter of 115±26 nm from Halothiobacillus neapolitanus. A new aerosol sample-injector allowed us to record 70,000 low-noise diffraction patterns in 12 min. Every diffraction pattern is a unique structure measurement and high-throughput imaging allows sampling the space of structural variability. The different structures can be separated and phased directly from the diffraction data and open a way for accurate, high-throughput studies on structures and structural heterogeneity in biology and elsewhere

Coherent diffraction of single Rice Dwarf virus particles using hard X-rays at the Linac Coherent Light Source

Single particle diffractive imaging data from Rice Dwarf Virus (RDV) were recorded using the Coherent X-ray Imaging (CXI) instrument at the Linac Coherent Light Source (LCLS). RDV was chosen as it is a wellcharacterized model system, useful for proof-of-principle experiments, system optimization and algorithm development. RDV, an icosahedral virus of about 70 nm in diameter, was aerosolized and injected into the approximately 0.1 mu m diameter focused hard X-ray beam at the CXI instrument of LCLS. Diffraction patterns from RDV with signal to 5.9 angstrom ngstrom were recorded. The diffraction data are available through the Coherent X-ray Imaging Data Bank (CXIDB) as a resource for algorithm development, the contents of which are described here.11Ysciescopu

Corporate governance and employees in South Africa.

Focusing on employees as stakeholders, we analyse corporate governance initiatives in South Africa encouraging and requiring companies to look beyond their shareholders' interests. Successive non-binding codes and the provisions of the recent Companies Act 2008 promoting this have been lauded by many commentators. The 2008 Act provides certain opportunities for employees and their representatives to exercise influence at the margins. We nevertheless question how far current corporate governance initiatives are adequate to promote employee interests. On the basis of three case studies of how companies have responded to employees as stakeholders, we conclude that in fact more stringent regulation is required

Prevalence and long-term change in alcohol consumption: results from a population-based cohort in Southern India

Background: Alcohol consumption in India is below the global average, with limited data on long-term effects. The current study aims to examine changes over time among alcohol consumers, the pattern of drinking and help-seeking for alcohol problems among South Indian men. Method: Data on the intake of various alcohol types were collected through standard questionnaires in two adult follow-ups [Baseline: 1998–2002, Follow-up: 2016–2019] from male participants in the Vellore birth cohort (VBC). Alcohol intake was converted to weekly standard drink units for analysis. Data on drinking patterns using the Alcohol Use Disorder Identification Test (AUDIT) and information on help-seeking among problem drinkers were collected during follow-up. Socio-demographic associations with alcohol consumption were determined using logistic regression. Results: The prevalence of alcohol consumption was 54.5% and 47.7% at the baseline and follow-up, respectively. Over two decades, 12% of men reported to have newly started drinking and 18% quit drinking. Lower education and lower socio-economic status (SES) were the strongest predictors of alcohol consumption. The AUDIT assessment among drinkers reported hazardous drinking of 38.4%, harmful drinking of 4.7% and 3.7% probable alcohol dependence. Among the persons with high AUDIT scores, 25% were concerned about high consumption, and 9% sought help to stop their alcohol consumption. Conclusion: Our results showed a decline in alcohol consumption in this cohort over two decades. Among drinkers, a high proportion report hazardous and harmful consumption. Low levels of education and SES are significant predictors of alcohol consumption. A low proportion of help-seeking reflects alcohol-related stigma in the community

Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser

Citation: Ekeberg, T., Svenda, M., Seibert, M. M., Abergel, C., Maia, F. R. N. C., Seltzer, V., . . . Hajdu, J. (2016). Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser. Scientific Data, 3. doi:10.1038/sdata.2016.60Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms