USP4 Auto-Deubiquitylation Promotes Homologous Recombination.

Repair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.Research in the S.P.J. laboratory is funded by CRUK Program Grant C6/A11224, CRUK Project Grant C6/A14831 and the European Community Seventh Framework Program grant agreement no. HEALTH-F2-2010-259893 (DDResponse). R.N. was funded by the Daiichi Sankyo Foundation of Life Sciences fellowship. Cancer Research UK Grant C6946/A14492 and Wellcome Trust Grant WT092096 provided core infrastructure funding. S.P.J receives his salary from the University of Cambridge, supplemented by CRUK. The John Fell Fund 133/075 and the Wellcome Trust grant 097813/Z/11/Z funded research performed by B.M.K and R.K..This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.molcel.2015.09.01

Human platelet protein ubiquitylation and changes following GPVI activation

Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibits platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. 925 of these peptides show an increase of more than 2-fold following stimulation with CRP-XL. Multiple sites of ubiquitylation were 16 identified on a number of proteins including Syk, filamin and integrin heterodimer subunits. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery

Colorectal cancer liver metastatic growth depends on PAD4-driven citrullination of the extracellular matrix

Citrullination of proteins, a post-translational conversion of arginine residues to citrulline, is recognized in rheumatoid arthritis, but largely undocumented in cancer. Here we show that citrullination of the extracellular matrix by cancer cell derived peptidylarginine deiminase 4 (PAD4) is essential for the growth of liver metastases from colorectal cancer (CRC). Using proteomics, we demonstrate that liver metastases exhibit higher levels of citrullination and PAD4 than unaffected liver, primary CRC or adjacent colonic mucosa. Functional significance for citrullination in metastatic growth is evident in murine models where inhibition of citrullination substantially reduces liver metastatic burden. Additionally, citrullination of a key matrix component collagen type I promotes greater adhesion and decreased migration of CRC cells along with increased expression of characteristic epithelial markers, suggesting a role for citrullination in promoting mesenchymal-to-epithelial transition and liver metastasis. Overall, our study reveals the potential for PAD4-dependant citrullination to drive the progression of CRC liver metastasis

Identification of a Phytase Gene in Barley (Hordeum vulgare L.)

Background: Endogenous phytase plays a crucial role in phytate degradation and is thus closely related to nutrient efficiency in barley products. The understanding of genetic information of phytase in barley can provide a useful tool for breeding new barley varieties with high phytase activity. Methodology/Principal Findings: Quantitative trait loci (QTL) analysis for phytase activity was conducted using a doubled haploid population. Phytase protein was purified and identified by the LC-ESI MS/MS Shotgun method. Purple acid phosphatase (PAP) gene was sequenced and the position was compared with the QTL controlling phytase activity. A major QTL for phytase activity was mapped to chromosome 5 H in barley. The gene controlling phytase activity in the region was named as mqPhy. The gene HvPAP a was mapped to the same position as mqPhy, supporting the colinearity between HvPAP a and mqPhy. Conclusions/Significance: It is the first report on QTLs for phytase activity and the results showed that HvPAP a, which shares a same position with the QTL, is a major phytase gene in barley grains

Enzymatic Sialylation of Synthetic Multivalent Scaffolds: From 3′-Sialyllactose Glycomacromolecules to Novel Neoglycosides

Sialoglycans play a key role in many biological recognition processes and sialylated conjugates of various types have successfully been applied, e.g., as antivirals or in antitumor therapy. A key feature for high affinity binding of such conjugates is the multivalent presentation of sialoglycans which often possess synthetic challenges. Here, the combination is described of solid phase polymer synthesis and enzymatic sialylation yielding 3′-sialyllactose-presenting precision glycomacromolecules. CMP-Neu5Ac synthetase from Neisseria meningitidis (NmCSS) and sialyltransferase from Pasteurella multocida (PmST1) are combined in a one-pot reaction giving access to sequence-defined sialylated macromolecules. Surprisingly, when employing Tris(hydroxymethyl)aminomethane (Tris) as a buffer, formation of significant amounts of α-linked Tris-sialoside is observed as a side reaction. Further exploring and exploiting this unusual sialylation reaction, different neoglycosidic structures are synthesized showing that PmST1 can be used to derive both, sialylation on natural carbohydrates as well as on synthetic hydroxylated scaffolds

Age influence on effectiveness of a novel 3-phytase in barley-wheat based diets for pigs from 12 to 108 kg under commercial conditions

[EN] The main objective of this study was to evaluate the influence of pig's age on the effectiveness of a new microbial 3-phytase, produced by Komagataella phaffii, under commercial conditions in barley-wheat based diets. Two experiments were conducted in weaned, growing and finishing pigs; firstly, to determine phytase efficacy on dry matter, organic matter, energy, protein and mineral (phosphorus, P and calcium, Ca) digestibility (n = 48; Experiment 1), and secondly, to evaluate the effect of phytase on growth performance and bone mineralization (n = 312; Experiment 2). In each experiment, three barley-wheat based diets were formulated following the recommendations for each animal age, of which two versions were manufactured, including 0 and 1000 phytase units (FTU)/kg of feed of the new 3-phytase to be tested. Results showed the new phytase had the potential to increase the digestibility of Ca and P (on av. + 0.05 and +0.06, respectively; P < 0.01), especially P digestibility in growing pigs (+0.10; P < 0.001), consequently decreasing P and Ca excretion. Digestible energy (DE) of the diet increased with the addition of phytase in weaned pigs (+0.69 MJ/kg of dry matter (DM); P < 0.001). Dietary inclusion of new 3-phytase enhanced average daily gain from 46 to 94 days of age (+0.07 kg/d; P < 0.05) and decreased feed conversion ratio from 46 to 154 days of age (on av. -0.13; P < 0.05), although no significant effect was observed from 154 to 185 days of age. Addition of the new 3-phytase also promoted bone mineralization, increasing the weight of the bones (+3.99 and +3.64 g of tibia at 95 days and metacarpus at 100 days of age, respectively; P < 0.05) and the ash, Ca and P content in these bones (e.g. + 0.46 and +0.33 g of P in tibia at 95 days and metacarpus at 100 days of age, respectively; P < 0.001). In conclusion, pig age affected the efficacy of a new 3-phytase on P and Ca digestibility both in weaned and growing diets and DE content of the weaned diets, which also resulted in improvements in growth, feed conversion and bone development until 154 days of age. These effects seem to be reduced during the finishing period, although the advantages of the new 3-phytase on bone mineralization were maintained until 185 days of age.We thank the technical staff at the experimental farms of the Research and Technology Animal Centre (CITA-IVIA), the Institute of Animal Science and Technology (Universitat Politècnica de Valencia) and Javier Gómez (Crianzas Campovivo) for expert technical assistance and experimental support.Cambra López, M.; Cerisuelo, A.; Ferrer, P.; Ródenas Martínez, L.; Aligué, R.; Moset, V.; Pascual Amorós, JJ. (2020). Age influence on effectiveness of a novel 3-phytase in barley-wheat based diets for pigs from 12 to 108 kg under commercial conditions. Animal Feed Science and Technology. 267:1-13. https://doi.org/10.1016/j.anifeedsci.2020.114549S113267Adeola, O., & Cowieson, A. J. (2011). BOARD-INVITED REVIEW: Opportunities and challenges in using exogenous enzymes to improve nonruminant animal production. Journal of Animal Science, 89(10), 3189-3218. doi:10.2527/jas.2010-3715Almeida, F. N., Sulabo, R. C., & Stein, H. H. (2013). Effects of a novel bacterial phytase expressed in Aspergillus Oryzae on digestibility of calcium and phosphorus in diets fed to weanling or growing pigs. Journal of Animal Science and Biotechnology, 4(1). doi:10.1186/2049-1891-4-8Arredondo, M. A., Casas, G. A., & Stein, H. H. (2019). Increasing levels of microbial phytase increases the digestibility of energy and minerals in diets fed to pigs. Animal Feed Science and Technology, 248, 27-36. doi:10.1016/j.anifeedsci.2019.01.001Atakora, J. K. A., Moehn, S., Sands, J. S., & Ball, R. O. (2011). Effects of dietary crude protein and phytase–xylanase supplementation of wheat grain based diets on energy metabolism and enteric methane in growing finishing pigs. Animal Feed Science and Technology, 166-167, 422-429. doi:10.1016/j.anifeedsci.2011.04.030Blaabjerg, K., Nørgaard, J. V., & Poulsen, H. D. (2012). Effect of microbial phytase on phosphorus digestibility in non-heat-treated and heat-treated wheat–barley pig diets1. Journal of Animal Science, 90(suppl_4), 206-208. doi:10.2527/jas.53920Brady, S., Callan, J., Cowan, D., McGrane, M., & O’Doherty, J. (2002). Effect of phytase inclusion and calcium/phosphorus ratio on the performance and nutrient retention of grower-finisher pigs fed barley/wheat/soya bean meal-based diets. Journal of the Science of Food and Agriculture, 82(15), 1780-1790. doi:10.1002/jsfa.1262Braña, D. V., Ellis, M., Castañeda, E. O., Sands, J. S., & Baker, D. H. (2006). Effect of a novel phytase on growth performance, bone ash, and mineral digestibility in nursery and grower-finisher pigs. Journal of Animal Science, 84(7), 1839-1849. doi:10.2527/jas.2005-565Dersjant‐Li, Y., Awati, A., Schulze, H., & Partridge, G. (2014). Phytase in non‐ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. Journal of the Science of Food and Agriculture, 95(5), 878-896. doi:10.1002/jsfa.6998Eeckhout, W., & De Paepe, M. (1994). Total phosphorus, phytate-phosphorus and phytase activity in plant feedstuffs. Animal Feed Science and Technology, 47(1-2), 19-29. doi:10.1016/0377-8401(94)90156-2EMIOLA, A., AKINREMI, O., SLOMINSKI, B., & NYACHOTI, C. M. (2009). Nutrient utilization and manure P excretion in growing pigs fed corn-barley-soybean based diets supplemented with microbial phytase. Animal Science Journal, 80(1), 19-26. doi:10.1111/j.1740-0929.2008.00590.xGonzález-Vega, J. C., Walk, C. L., & Stein, H. H. (2015). Effects of microbial phytase on apparent and standardized total tract digestibility of calcium in calcium supplements fed to growing pigs1. Journal of Animal Science, 93(5), 2255-2264. doi:10.2527/jas.2014-8215Harper, A. F., Kornegay, E. T., & Schell, T. C. (1997). Phytase supplementation of low-phosphorus growing-finishing pig diets improves performance, phosphorus digestibility, and bone mineralization and reduces phosphorus excretion. Journal of Animal Science, 75(12), 3174. doi:10.2527/1997.75123174xHaug, W., & Lantzsch, H.-J. (1983). Sensitive method for the rapid determination of phytate in cereals and cereal products. Journal of the Science of Food and Agriculture, 34(12), 1423-1426. doi:10.1002/jsfa.2740341217Heaney, R. P., Abrams, S., Dawson-Hughes, B., Looker, A., Looker, A., Marcus, R., … Weaver, C. (2001). Peak Bone Mass. Osteoporosis International, 11(12), 985-1009. doi:10.1007/s001980070020Jørgensen, B. (1995). Effect of different energy and protein levels on leg weakness and osteochondrosis in pigs. Livestock Production Science, 41(2), 171-181. doi:10.1016/0301-6226(94)00048-cKemme, P. A., Jongbloed, A. W., Mroz, Z., & Beynen, A. C. (1997). The efficacy of Aspergillus niger phytase in rendering phytate phosphorus available for absorption in pigs is influenced by pig physiological status. Journal of Animal Science, 75(8), 2129. doi:10.2527/1997.7582129xKiela, P. R., & Ghishan, F. K. (2016). Physiology of Intestinal Absorption and Secretion. Best Practice & Research Clinical Gastroenterology, 30(2), 145-159. doi:10.1016/j.bpg.2016.02.007Kim, J. C., Simmins, P. H., Mullan, B. P., & Pluske, J. R. (2005). The effect of wheat phosphorus content and supplemental enzymes on digestibility and growth performance of weaner pigs. Animal Feed Science and Technology, 118(1-2), 139-152. doi:10.1016/j.anifeedsci.2004.08.016Koch, M. E., & Mahan, D. C. (1985). Biological Characteristics for Assessing Low Phosphorus Intake in Growing Swine. Journal of Animal Science, 60(3), 699-708. doi:10.2527/jas1985.603699xKonietzny, U., & Greiner, R. (2002). Molecular and catalytic properties of phytate-degrading enzymes (phytases). International Journal of Food Science and Technology, 37(7), 791-812. doi:10.1046/j.1365-2621.2002.00617.xLittell, R. C., Henry, P. R., & Ammerman, C. B. (1998). Statistical analysis of repeated measures data using SAS procedures. Journal of Animal Science, 76(4), 1216. doi:10.2527/1998.7641216xMahan, D. C. (1982). Dietary Calcium and Phosphorus Levels for Weanling Swine. Journal of Animal Science, 54(3), 559-564. doi:10.2527/jas1982.543559xMavromichalis, I., Hancock, J. D., Kim, I. H., Senne, B. W., Kropf, D. H., Kennedy, G. A., … Behnke, K. C. (1999). Effects of omitting vitamin and trace mineral premixes and(or) reducing inorganic phosphorus additions on growth performance, carcass characteristics, and muscle quality in finishing pigs. Journal of Animal Science, 77(10), 2700. doi:10.2527/1999.77102700xMoehn, S., Atakora, J. K. A., Sands, J., & Ball, R. O. (2007). Effect of phytase-xylanase supplementation to wheat-based diets on energy metabolism in growing–finishing pigs fed ad libitum. Livestock Science, 109(1-3), 271-274. doi:10.1016/j.livsci.2007.01.118Oryschak, M. A., Simmins, P. H., & Zijlstra, R. T. (2002). Effect of dietary particle size and carbohydrase and/or phytase supplementation on nitrogen and phosphorus excretion of grower pigs. Canadian Journal of Animal Science, 82(4), 533-540. doi:10.4141/a02-016Peter, C. ., Parr, T. ., Parr, E. ., Webel, D. ., & Baker, D. . (2001). The effects of phytase on growth performance, carcass characteristics, and bone mineralization of late-finishing pigs fed maize–soyabean meal diets containing no supplemental phosphorus, zinc, copper and manganese. Animal Feed Science and Technology, 94(3-4), 199-205. doi:10.1016/s0377-8401(01)00300-5Rodehutscord, M., Faust, M., & Lorenz, H. (1996). Digestibility of phosphorus contained in soybean meal, barley, and different varieties of wheat, without and with supplemental phytase fed to pigs and additivity of digestibility in a wheatsoybean-meal diet. Journal of Animal Physiology and Animal Nutrition, 75(1-5), 40-48. doi:10.1111/j.1439-0396.1996.tb00466.xSelle, P. H., & Ravindran, V. (2008). Phytate-degrading enzymes in pig nutrition. Livestock Science, 113(2-3), 99-122. doi:10.1016/j.livsci.2007.05.014Selle, P. H., Cadogan, D. J., & Bryden, W. L. (2003). Effects of phytase supplementation of phosphorus-adequate, lysine-deficient, wheat-based diets on growth performance of weaner pigs. Australian Journal of Agricultural Research, 54(3), 323. doi:10.1071/ar02121She, Y., Su, Y., Liu, L., Huang, C., Li, J., Li, P., … Piao, X. (2015). Effects of microbial phytase on coefficient of standardized total tract digestibility of phosphorus in growing pigs fed corn and corn co-products, wheat and wheat co-products and oilseed meals. Animal Feed Science and Technology, 208, 132-144. doi:10.1016/j.anifeedsci.2015.07.011Van Soest, P. J., Robertson, J. B., & Lewis, B. A. (1991). Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. Journal of Dairy Science, 74(10), 3583-3597. doi:10.3168/jds.s0022-0302(91)78551-2Varley, P. F., Callan, J. J., & O’Doherty, J. V. (2011). Effect of dietary phosphorus and calcium level and phytase addition on performance, bone parameters, apparent nutrient digestibility, mineral and nitrogen utilization of weaner pigs and the subsequent effect on finisher pig bone parameters. Animal Feed Science and Technology, 165(3-4), 201-209. doi:10.1016/j.anifeedsci.2011.02.017Varley, P. F., Flynn, B., Callan, J. J., & O’Doherty, J. V. (2011). Effect of phytase level in a low phosphorus diet on performance and bone development in weaner pigs and the subsequent effect on finisher pig bone development. Livestock Science, 138(1-3), 152-158. doi:10.1016/j.livsci.2010.12.014Vipperman, P. E., Peo, E. R., & Cunningham, P. J. (1974). Effect of Dietary Calcium and Phosphorus Level upon Calcium, Phosphorus and Nitrogen Balance in Swine. Journal of Animal Science, 38(4), 758-765. doi:10.2527/jas1974.384758

The Jumonji-C oxygenase JMJD7 catalyzes (3S)-lysyl hydroxylation of TRAFAC GTPases

Biochemical, structural and cellular studies reveal Jumonji-C (JmjC) domain-containing 7 (JMJD7) to be a 2-oxoglutarate (2OG)-dependent oxygenase that catalyzes (3S)-lysyl hydroxylation. Crystallographic analyses reveal JMJD7 to be more closely related to the JmjC hydroxylases than to the JmjC demethylases. Biophysical and mutation studies show that JMJD7 has a unique dimerization mode, with interactions between monomers involving both N- and C-terminal regions and disulfide bond formation. A proteomic approach identifies two related members of the translation factor (TRAFAC) family of GTPases, developmentally regulated GTP-binding proteins 1 and 2 (DRG1/2), as activity-dependent JMJD7 interactors. Mass spectrometric analyses demonstrate that JMJD7 catalyzes Fe(ii)- and 2OG-dependent hydroxylation of a highly conserved lysine residue in DRG1/2; amino-acid analyses reveal that JMJD7 catalyzes (3S)-lysyl hydroxylation. The functional assignment of JMJD7 will enable future studies to define the role of DRG hydroxylation in cell growth and disease.Fil: Markolovic, Suzana. University of Oxford; Reino UnidoFil: Zhuang, Qinqin. University Of Birmingham; Reino UnidoFil: Wilkins, Sarah E.. University of Oxford; Reino UnidoFil: Eaton, Charlotte D.. University Of Birmingham; Reino UnidoFil: Abboud, Martine I.. University of Oxford; Reino UnidoFil: Katz, Maximiliano Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: McNeil, Helen E.. University Of Birmingham; Reino UnidoFil: Leśniak, Robert K.. University of Oxford; Reino UnidoFil: Hall, Charlotte. University Of Birmingham; Reino UnidoFil: Struwe, Weston B.. University of Oxford; Reino UnidoFil: Konietzny, Rebecca. University of Oxford; Reino UnidoFil: Davis, Simon. University of Oxford; Reino UnidoFil: Yang, Ming. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Ge, Wei. University of Oxford; Reino UnidoFil: Benesch, Justin L. P.. University of Oxford; Reino UnidoFil: Kessler, Benedikt M.. University of Oxford; Reino UnidoFil: Ratcliffe, Peter J.. University of Oxford; Reino Unido. The Francis Crick Institute; Reino UnidoFil: Cockman, Matthew E.. The Francis Crick Institute; Reino Unido. University of Oxford; Reino UnidoFil: Fischer, Roman. University of Oxford; Reino UnidoFil: Wappner, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Chowdhury, Rasheduzzaman. University of Stanford; Estados Unidos. University of Oxford; Reino UnidoFil: Coleman, Mathew L.. University Of Birmingham; Reino UnidoFil: Schofield, Christopher J.. University of Oxford; Reino Unid