The de novo synthesis of ubiquitin: Identification of deubiquitinases acting on ubiquitin precursors

Protein ubiquitination, a major post-translational modification in eukaryotes, requires an adequate pool of free ubiquitin. Cells maintain this pool by two pathways, both involving deubiquitinases (DUBs): recycling of ubiquitin from ubiquitin conjugates and processing of ubiquitin precursors synthesized de novo. Although many advances have been made in recent years regarding ubiquitin recycling, our knowledge on ubiquitin precursor processing is still limited, and questions such as when are these precursors processed and which DUBs are involved remain largely unanswered. Here we provide data suggesting that two of the four mammalian ubiquitin precursors, UBA 52 and UBA 80 , are processed mostly post-translationally whereas the other two, UBB and UBC, probably undergo a combination of co-and post-translational processing. Using an unbiased biochemical approach we found that UCHL 3 , USP 9 X, USP 7 , USP 5 and Otulin/Gumby/FAM 105 b are by far the most active DUBs acting on these precursors. The identification of these DUBs together with their properties suggests that each ubiquitin precursor can be processed in at least two different manners, explaining the robustness of the ubiquitin de novo synthesis pathway.We are grateful to Dr. Cheryl Arrowsmith (University of Toronto, Canada) for providing the plasmids pET28a-LIC-USP5 (Addgene plasmid 25299) and pET28a-LIC-USP5(C335A). We thank Dr. João M. Cabral (IBMC, University of Porto, Portugal) for critically reading the manuscript. This work was supported by national funds through FCT - Fundação para a Ciência e a Tecnologia/MEC – Ministério da Educação e Ciência and when applicable co-funded by Fundo de Desenvolvimento Regional (FEDER) funds within the partnership agreement PT2020 related with the research unit number 4293; by Project “NORTE-07-0124-FEDER-000003 -Cell homeotasis tissue organization and organism biology”, co-funded by Programa Operacional Regional do Norte (ON.2—O Novo Norte), under the Quadro de Referência Estratégico Nacional (QREN), through FEDER and by FCT; by Portuguese National Mass Spectrometry Network (RNEM) through the project REDE/1504/REM/2005; and by Química Orgânica, Produtos Naturais e Agroalimentares (QOPNA) research unit funds provided by FCT, European Union, QREN, FEDER and Operational Competitiveness Programme (COMPETE) under the projects PEst-C/QUI/UI0062/2013 and FCOMP-01-0124-FEDER-037296. C.P.G. and M.P.P. were supported by FCT, COMPETE and Fundo Social Europeu. A.V.M. was supported by the project FCOMP-01-0124-FEDER-027627-EXPL/BEX-BCM/0320/2012 financed by national funds from FCT/Ministério da Educação e Ciência (PIDDAC) and co-funded by FEDER through COMPETE—Programa Operacional Factores de Competitividade (POFC)

Model-Derived Dispersal Pathways from Multiple Source Populations Explain Variability of Invertebrate Larval Supply

Background: Predicting the spatial and temporal patterns of marine larval dispersal and supply is a challenging task due to the small size of the larvae and the variability of oceanographic processes. Addressing this problem requires the use of novel approaches capable of capturing the inherent variability in the mechanisms involved. Methodology/Principal Findings: In this study we test whether dispersal and connectivity patterns generated from a biophysical model of larval dispersal of the crab Carcinus maenas, along the west coast of the Iberian Peninsula, can predict the highly variable daily pattern of wind-driven larval supply to an estuary observed during the peak reproductive season (March–June) in 2006 and 2007. Cross-correlations between observed and predicted supply were significant (p,0.05) and strong, ranging from 0.34 to 0.81 at time lags of 26 to+5 d. Importantly, the model correctly predicted observed cross-shelf distributions (Pearson r = 0.82, p,0.001, and r = 0.79, p,0.01, in 2006 and 2007) and indicated that all supply events were comprised of larvae that had been retained within the inner shelf; larvae transported to the outer shelf and beyond never recruited. Estimated average dispersal distances ranged from 57 to 198 km and were only marginally affected by mortality. Conclusions/Significance: The high degree of predicted demographic connectivity over relatively large geographic scales is consistent with the lack of genetic structuring in C. maenas along the Iberian Peninsula. These findings indicate that the dynamic nature of larval dispersal can be captured by mechanistic biophysical models, which can be used to provid

Expansion of oxygen minimum zones may reduce available habitat for tropical pelagic fishes

Climate model predictions1, 2 and observations3, 4 reveal regional declines in oceanic dissolved oxygen, which are probably influenced by global warming5. Studies indicate ongoing dissolved oxygen depletion and vertical expansion of the oxygen minimum zone (OMZ) in the tropical northeast Atlantic Ocean6, 7. OMZ shoaling may restrict the usable habitat of billfishes and tunas to a narrow surface layer8, 9. We report a decrease in the upper ocean layer exceeding 3.5 ml l−1 dissolved oxygen at a rate of ≤1 m yr−1 in the tropical northeast Atlantic (0–25° N, 12–30° W), amounting to an annual habitat loss of ~5.95×1013 m3, or 15% for the period 1960–2010. Habitat compression and associated potential habitat loss was validated using electronic tagging data from 47 blue marlin. This phenomenon increases vulnerability to surface fishing gear for billfishes and tunas8, 9, and may be associated with a 10–50% worldwide decline of pelagic predator diversity10. Further expansion of the Atlantic OMZ along with overfishing may threaten the sustainability of these valuable pelagic fisheries and marine ecosystems

Making sense of the early-2000s warming slowdown

It has been claimed that the early-2000s global warming slowdown or hiatus, characterized by a reduced rate of global surface warming, has been overstated, lacks sound scientific basis, or is unsupported by observations. The evidence presented here contradicts these claims

Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4- methylumbelliferyl-β-D-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.Fundação para a Ciência e a Tecnologia (FCT

Reconciling Deep Calibration and Demographic History: Bayesian Inference of Post Glacial Colonization Patterns in Carcinus aestuarii (Nardo, 1847) and C. maenas (Linnaeus, 1758)

A precise inference of past demographic histories including dating of demographic events using Bayesian methods can only be achieved with the use of appropriate molecular rates and evolutionary models. Using a set of 596 mitochondrial cytochrome c oxidase I (COI) sequences of two sister species of European green crabs of the genus Carcinus (C. maenas and C. aestuarii), our study shows how chronologies of past evolutionary events change significantly with the application of revised molecular rates that incorporate biogeographic events for calibration and appropriate demographic priors. A clear signal of demographic expansion was found for both species, dated between 10,000 and 20,000 years ago, which places the expansions events in a time frame following the Last Glacial Maximum (LGM). In the case of C. aestuarii, a population expansion was only inferred for the Adriatic-Ionian, suggestive of a colonization event following the flooding of the Adriatic Sea (18,000 years ago). For C. maenas, the demographic expansion inferred for the continental populations of West and North Europe might result from a northward recolonization from a southern refugium when the ice sheet retreated after the LGM. Collectively, our results highlight the importance of using adequate calibrations and demographic priors in order to avoid considerable overestimates of evolutionary time scales

A reporting format for leaf-level gas exchange data and metadata

Leaf-level gas exchange data support the mechanistic understanding of plant fluxes of carbon and water. These fluxes inform our understanding of ecosystem function, are an important constraint on parameterization of terrestrial biosphere models, are necessary to understand the response of plants to global environmental change, and are integral to efforts to improve crop production. Collection of these data using gas analyzers can be both technically challenging and time consuming, and individual studies generally focus on a small range of species, restricted time periods, or limited geographic regions. The high value of these data is exemplified by the many publications that reuse and synthesize gas exchange data, however the lack of metadata and data reporting conventions make full and efficient use of these data difficult. Here we propose a reporting format for leaf-level gas exchange data and metadata to provide guidance to data contributors on how to store data in repositories to maximize their discoverability, facilitate their efficient reuse, and add value to individual datasets. For data users, the reporting format will better allow data repositories to optimize data search and extraction, and more readily integrate similar data into harmonized synthesis products. The reporting format specifies data table variable naming and unit conventions, as well as metadata characterizing experimental conditions and protocols. For common data types that were the focus of this initial version of the reporting format, i.e., survey measurements, dark respiration, carbon dioxide and light response curves, and parameters derived from those measurements, we took a further step of defining required additional data and metadata that would maximize the potential reuse of those data types. To aid data contributors and the development of data ingest tools by data repositories we provided a translation table comparing the outputs of common gas exchange instruments. Extensive consultation with data collectors, data users, instrument manufacturers, and data scientists was undertaken in order to ensure that the reporting format met community needs. The reporting format presented here is intended to form a foundation for future development that will incorporate additional data types and variables as gas exchange systems and measurement approaches advance in the future. The reporting format is published in the U.S. Department of Energy's ESS-DIVE data repository, with documentation and future development efforts being maintained in a version control system

Design Constraints on a Synthetic Metabolism

A metabolism is a complex network of chemical reactions that converts sources of energy and chemical elements into biomass and other molecules. To design a metabolism from scratch and to implement it in a synthetic genome is almost within technological reach. Ideally, a synthetic metabolism should be able to synthesize a desired spectrum of molecules at a high rate, from multiple different nutrients, while using few chemical reactions, and producing little or no waste. Not all of these properties are achievable simultaneously. We here use a recently developed technique to create random metabolic networks with pre-specified properties to quantify trade-offs between these and other properties. We find that for every additional molecule to be synthesized a network needs on average three additional reactions. For every additional carbon source to be utilized, it needs on average two additional reactions. Networks able to synthesize 20 biomass molecules from each of 20 alternative sole carbon sources need to have at least 260 reactions. This number increases to 518 reactions for networks that can synthesize more than 60 molecules from each of 80 carbon sources. The maximally achievable rate of biosynthesis decreases by approximately 5 percent for every additional molecule to be synthesized. Biochemically related molecules can be synthesized at higher rates, because their synthesis produces less waste. Overall, the variables we study can explain 87 percent of variation in network size and 84 percent of the variation in synthesis rate. The constraints we identify prescribe broad boundary conditions that can help to guide synthetic metabolism design