Dynamic control of selectivity in the ubiquitination pathway revealed by an ASP to GLU substitution in an intra-molecular salt-bridge network

Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity

Promiscuous, non-catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and development of transgenic tobacco (Nicotiana tabacum) plants

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth

Limited Effect of Y Chromosome Variation on Coronary Artery Disease and Mortality in UK Biobank

The effect of genetic variation in the male-specific region of the Y chromosome (MSY) on coronary artery disease and cardiovascular risk factors has been disputed. In this study, we systematically assessed the association of MSY genetic variation on these traits using a kin-cohort analysis of family disease history in the largest sample to date. METHODS: We tested 90 MSY haplogroups against coronary artery disease, hypertension, blood pressure, classical lipid levels, and all-cause mortality in up to 152 186 unrelated, genomically British individuals from UK Biobank. Unlike previous studies, we did not adjust for heritable lifestyle factors (to avoid collider bias) and instead adjusted for geographic variables and socioeconomic deprivation, given the link between MSY haplogroups and geography. For family history traits, subject MSY haplogroups were tested against father and mother disease as validation and negative control, respectively. RESULTS: Our models find little evidence for an effect of any MSY haplogroup on cardiovascular risk in participants. Parental models confirm these findings. CONCLUSIONS: Kin-cohort analysis of the Y chromosome uniquely allows for discoveries in subjects to be validated using family history data. Despite our large sample size, improved models, and parental validation, there is little evidence to suggest cardiovascular risk in UK Biobank is influenced by genetic variation in MSY

The use of biotin tagging in Saccharomyces cerevisiae improves the sensitivity of chromatin immunoprecipitation

Affinity tagging has been used in many global studies towards protein function. We describe a highly efficient system for in vivo biotinylation of transcription factors in the yeast Saccharomyces cerevisiae, which is based on the bacterial BirA biotin ligase. The strength of the biotin–streptavidin interaction was exploited to improve detection of in vivo protein–DNA complexes in chromatin immunoprecipitation (ChIP) experiments. In a test system using the biotin-tagged LexA DNA-binding protein, we found that stringent washing conditions resulted in a strong improvement of the signal-to-noise ratios. Yeast strains with chromosomally integrated versions of tagged transcription factor genes were generated using N- or C-terminal biotin-tagging cassettes. ChIP experiments with biotinylated Rbp3p, a RNA polymerase II subunit, showed that Rbp3p-binding could even be detected at weakly expressed genes. Other methods failed to detect RNA polymerase II binding at such genes. Our results show that biotinylation of yeast transcription factors improves the detection of in vivo protein–DNA complexes

Evidence of Double Phonon Excitations in ^{16}O + ^{208}Pb Reaction

The fusion cross-sections for ^{16}O + ^{208}Pb, measured to high precision, enable the extraction of the distribution of fusion barriers. This shows a structure markedly different from the single-barrier which might be expected for fusion of two doubly-closed shell nuclei. The results of exact coupled channel calculations performed to understand the observations are presented. These calculations indicate that coupling to a double octupole phonon excited state in ^{208}Pb is necessary to explain the experimental barrier distributions.Comment: 6 pages, 2 figures, To be published in the Proceedings of the FUSION 97 Conference, South Durras, Australia, March 1997 (J. Phys. G

Reverse methanogenesis and respiration in methanotrophic archaea

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., "trace methane oxidation"). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.The authors thank Stefanie Berger (RU,Nijmegen) for critical reading of the manuscript. This research is supported by the Soehngen Institute of Anaerobic Microbiology (SIAM) Gravitation Grant (024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Organisation for Scientific Research (NWO). Mike S. M. Jetten was further supported by ERC AG 339880 Eco-MoM and Alfons J. M. Stams was supported by ERC AG 323009 Novel Anaerobes.info:eu-repo/semantics/publishedVersio

Metabolism and occurrence of methanogenic and sulfate-reducing syntrophic acetate oxidizing communities in haloalkaline environments

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.03039/full#supplementary-materialAnaerobic syntrophic acetate oxidation (SAO) is a thermodynamically unfavorable process involving a syntrophic acetate oxidizing bacterium (SAOB) that forms interspecies electron carriers (IECs). These IECs are consumed by syntrophic partners, typically hydrogenotrophic methanogenic archaea or sulfate reducing bacteria. In this work, the metabolism and occurrence of SAOB at extremely haloalkaline conditions were investigated, using highly enriched methanogenic (M-SAO) and sulfate-reducing (S-SAO) cultures from south-eastern Siberian hypersaline soda lakes. Activity tests with the M-SAO and S-SAO cultures and thermodynamic calculations indicated that hydrogen and formate are important IECs in both SAO cultures. Metagenomic analysis of the M-SAO cultures showed that the dominant SAOB was Candidatus Syntrophonatronum acetioxidans, and a near-complete draft genome of this SAOB was reconstructed. Ca. S. acetioxidans has all genes necessary for operating the Wood-Ljungdahl pathway, which is likely employed for acetate oxidation. It also encodes several genes essential to thrive at haloalkaline conditions; including a Na+-dependent ATP synthase and marker genes for salt-out strategies for osmotic homeostasis at high soda conditions. Membrane lipid analysis of the M-SAO culture showed the presence of unusual bacterial diether membrane lipids which are presumably beneficial at extreme haloalkaline conditions. To determine the importance of SAO in haloalkaline environments, previously obtained 16S rRNA gene sequencing data and metagenomic data of five different hypersaline soda lake sediment samples were investigated, including the soda lakes where the enrichment cultures originated from. The draft genome of Ca. S. acetioxidans showed highest identity with two metagenome-assembled genomes (MAGs) of putative SAOBs that belonged to the highly abundant and diverse Syntrophomonadaceae family present in the soda lake sediments. The 16S rRNA amplicon datasets of the soda lake sediments showed a high similarity of reads to Ca. S. acetioxidans with abundance as high as 1.3% of all reads, whereas aceticlastic methanogens and acetate oxidizing sulfate-reducers were not abundant (0.1%) or could not be detected. These combined results indicate that SAO is the primary anaerobic acetate oxidizing pathway at extreme haloalkaline conditions performed by haloalkaliphilic syntrophic consortia.This research was supported by the Soehngen Institute of AnaerobicMicrobiology(SIAM) Gravitation grant(024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Organisation for Scientific Research (NWO). GM and CV were supported by the ERC Advanced Grant PARASOL (No. 322551). DS also received support from the Russian Foundation for Basic Research (16-04-00035) and the Russian Academy of Sciences and Federal Agency of Scientific Organizations(0104-2018-0033), AS by the ERC Advanced Grant Novel Anaerobes (No. 323009), and JD by the ERC Advanced Grant Microlipids (No.694569).info:eu-repo/semantics/publishedVersio

Volumetric evaluation of CT images of adrenal glands in primary aldosteronism

Objectives: To investigate whether adrenal volumetry provides better agreement with adrenal vein sampling (AVS) than conventional CT for subtyping PA. Furthermore, we evaluated whether the size of this contralateral adrenal was a prognostic factor for clinical outcome after unilateral adrenalectomy.Methods: We retrospectively analyzed volumes of both adrenal glands of the 180 CT-scans (88/180 with unilateral and 92/180 with bilateral disease) of the patients with PA included in the SPARTACUS trial of which 85 also had undergone an AVS. In addition, we examined CT-scans of 20 healthy individuals to compare adrenal volumes with published normal values.Results: Adrenal volume was higher for the left than the right adrenal (mean and SD: 6.49 ± 2.77 ml versus 5.25 ± 1.87 ml for the right adrenal; p &lt; 0.001). Concordance between volumetry and AVS in subtyping was 58.8%, versus 51.8% between conventional CT results and AVS (p = NS). The volumes of the contralateral adrenals in the patients with unilateral disease (right 4.78 ± 1.37 ml; left 6.00 ± 2.73 ml) were higher than those of healthy controls reported in the literature (right 3.62 ± 1.23 ml p &lt; 0.001; left 4.84 ± 1.67 ml p = 0.02). In a multivariable analysis the contralateral volume was not associated with biochemical or clinical success, nor with the defined daily doses of antihypertensive agents at 1 year follow-up.Conclusions: Volumetry of the adrenal glands is not superior to current assessment of adrenal size by CT for subtyping patients with PA. Furthermore, in patients with unilateral disease the size of the contralateral adrenal is enlarged but its size is not associated with outcome.</p

Nitrogen-rich indium nitride

Elastic recoil detection analysis, using an incident beam of 200 MeV Au ions, has been used to measureindium nitride films grown by radio-frequency sputtering. It is shown that the films have nitrogen-rich stoichiometry. Nitrogen vacancies are therefore unlikely to be responsible for the commonly observed high background carrier concentration. Ultraviolet Raman and secondary ion mass spectroscopymeasurements are used to probe the state of the excess nitrogen. The nitrogen on indium anti-site defect is implicated, though other possibilities for the site of the excess nitrogen, such as molecular nitrogen, or di-nitrogen interstitials cannot be excluded. It is further shown that a shift in the (0002) x-ray diffraction peak correlates with the excess nitrogen, but not with the oxygen observed in some samples.K.S.A.B. would like to acknowledge the support of an Australian Research Council Fellowship. We would also like to acknowledge the support of the Australian Research Council through a Large grant and a Discovery grant; the support of a Macquarie University Research Development Grant, and the Australian Institute of Nuclear Science and Engineering for SIMS access