Automatic Assignment of EC Numbers

A wide range of research areas in molecular biology and medical biochemistry require a reliable enzyme classification system, e.g., drug design, metabolic network reconstruction and system biology. When research scientists in the above mentioned areas wish to unambiguously refer to an enzyme and its function, the EC number introduced by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB) is used. However, each and every one of these applications is critically dependent upon the consistency and reliability of the underlying data for success. We have developed tools for the validation of the EC number classification scheme. In this paper, we present validated data of 3788 enzymatic reactions including 229 sub-subclasses of the EC classification system. Over 80% agreement was found between our assignment and the EC classification. For 61 (i.e., only 2.5%) reactions we found that their assignment was inconsistent with the rules of the nomenclature committee; they have to be transferred to other sub-subclasses. We demonstrate that our validation results can be used to initiate corrections and improvements to the EC number classification scheme.     

Low genetic and morphological differentiation in the European species complex of Rosa sherardii,R. mollis and R. villosa (Rosa section Caninae subsection Vestitae)

The polyploid species complex of Rosa villosa sensu lato (Rosa section Caninae subsection Vestitae) consists of three morphologically similar polyploid species: R. sherardii, R. mollis and R. villosa. Whereas R. sherardii is distributed in central Europe, R. mollis and R. villosa represent a vicariant species pair occurring in northern Europe and in mountains of central and eastern Europe, respectively. In this study we analysed multiple data sets (morphology, cytology, microsatellites, AFLP and plastid DNA sequences) to re‐evaluate the systematics of these species and to examine whether cytological differences are reflected in the taxonomy and geographical distributions. Furthermore, these data sets were used to evaluate hypotheses explaining the vicariant distribution of R. mollis and R. villosa. None of these data sets revealed a clear‐cut differentiation between the species. Cytological and molecular data argued for a discrete taxonomic position of the predominantly pentaploid R. sherardii, but these data did not support a separation between the mostly tetraploid R. mollis and R. villosa. Population genetics revealed that samples of the latter species were assembled according to ploidy, but not to species affiliation or geographical distribution. Thus, we assume that the cytologically polymorphic original species had a continuous range prior to the last glaciation period and survived on nunataks or in non‐glaciated coastal regions in northern Europe, but that it failed to recover its former range after the retreat of the ice sheets. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 174 , 240–256.     

Initial phases of gravity-induced lateral auxin transport and geotropic curvature in corn coleoptiles

Summary Wild-type corn coleoptiles showed an initial downward bending upon transfer from the vertical to the horizontal position. Strong upward curvature started only 15–30 min after the begin of horizontal exposure.Little, if any at all, initial downward geotropic bending was found with amylomaize coleoptiles at 1 X g. With stronger stimuli (10 or 20 X g) the amylomaize mutant reacted initially strongly in the wrong direction, i.e. opposite to the later response.When wild-type coleoptiles had been symmetrically prestimulated for 60 min with alternating 2-min horizontal exposures from opposite sides, no initial downward bending occurred if the plane of horizontal exposure was maintained from pretreatment to the continuous horizontal stimulation of the test. If, however, the coleoptiles were rotated 90° around their long axis between pretreatment and test, the initial downward bending reaction developed as in the non-prestimulated controls. Thus changes in reactivity remained localized to the site of stimulation.Following the same pretreatments used for the curvature measurements, lateral ³H-IAA transport was measured in coleoptile segments for 10 or 12.5 min. The auxin distribution found was strikingly parallel to the bending for all pretreatments.The dependence of reaction pattern on the duration of prestimulation in the same plane was tested. The function indicates a half life of 10–20 min for the change in sensitivity. The findings are discussed in view of a model of overstimulation and adaptation.     

Prolonged culture of HOS 58 human osteosarcoma cells with 1,25-(OH)2-D3, TGF-beta, and dexamethasone reveals physiological regulation of alkaline phosphatase, dissociated osteocalcin gene expression, and protein synthesis and lack of mineralization

Cultured rodent osteoblastic cells reiterate the phenotypic differentiation and maturation of osteoblasts seen in vivo. As previously shown, the human osteosarcoma cell line HOS 58 represents a differentiated stage of osteoblast development. The potential of HOS 58 for still further in vitro differentiation suggests the line can serve as a model of osteoblast maturation. Using this cell line, we have investigated the influence of 1,25-(OH)2-D3 (D3), TGF-beta and Dexamethasone (Dex) on proliferation and on the protein and mRNA levels of alkaline phosphatase (AP), procollagen 1 (Col 1), and osteocalcin (Oc), as well as mineralization during 28 days in culture. AP mRNA and protein were highly expressed throughout the culture period with further increase of protein AP activity at constant gene expression levels. A differentiation inhibiting effect of either TGF-beta or Dex was seen. Col 1 was investigated without the use of ascorbic acid and showed only minor changes during culture time or stimulation. The gene expression for Oc increased continually whereas protein synthesis peaked at confluence and decreased thereafter. TGF-beta and Dex treatments decreased Oc mRNA and protein levels. Stimulation by D3 was maximal at day 7 with a decrease thereafter. HOS 58 cells showed no mineralization capacity when stimulated with different agents, as measured by energy-dispersive X-ray microanalysis. This was not due to absence of Cbfa1 expression. In conclusion, the HOS 58 osteosarcoma cell line represents a differentiated cell line with highly expressed and physiologically regulated AP expression during further differentiation in culture. We observed a dissociation between osteocalcin gene expression and protein secretion which may contribute to the lack of mineralization in this cell line.     

Maintenance of C sinks sustains enhanced C assimilation during long-term exposure to elevated [CO2] in Mojave Desert shrubs

During the first few years of elevated atmospheric [CO(2)] treatment at the Nevada Desert FACE Facility, photosynthetic downregulation was observed in desert shrubs grown under elevated [CO(2)], especially under relatively wet environmental conditions. Nonetheless, those plants maintained increased A (sat) (photosynthetic performance at saturating light and treatment [CO(2)]) under wet conditions, but to a much lesser extent under dry conditions. To determine if plants continued to downregulate during long-term exposure to elevated [CO(2)], responses of photosynthesis to elevated [CO(2)] were examined in two dominant Mojave Desert shrubs, the evergreen Larrea tridentata and the drought-deciduous Ambrosia dumosa, during the eighth full growing season of elevated [CO(2)] treatment at the NDFF. A comprehensive suite of physiological processes were collected. Furthermore, we used C labeling of air to assess carbon allocation and partitioning as measures of C sink activity. Results show that elevated [CO(2)] enhanced photosynthetic performance and plant water status in Larrea, especially during periods of environmental stress, but not in Ambrosia. δ(13)C analyses indicate that Larrea under elevated [CO(2)] allocated a greater proportion of newly assimilated C to C sinks than Ambrosia. Maintenance by Larrea of C sinks during the dry season partially explained the reduced [CO(2)] effect on leaf carbohydrate content during summer, which in turn lessened carbohydrate build-up and feedback inhibition of photosynthesis. δ(13)C results also showed that in a year when plant growth reached the highest rates in 5 years, 4% (Larrea) and 7% (Ambrosia) of C in newly emerging organs were remobilized from C that was assimilated and stored for at least 2 years prior to the current study. Thus, after 8 years of continuous exposure to elevated [CO(2)], both desert perennials maintained their photosynthetic capacities under elevated [CO(2)]. We conclude that C storage, remobilization, and partitioning influence the responsiveness of these desert shrubs during long-term exposure to elevated [CO(2)].     

Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells

The initial response of renal epithelial cells to the antidiuretic hormone arginine vasopressin (AVP) is an increase in cyclic AMP. By applying immunofluorescence, cell membrane capacitance and transepithelial water flux measurements we show that cAMP alone is sufficient to elicit the antidiuretic cellular response in primary cultured epithelial cells from renal inner medulla, namely the transport of aquaporin-2 (AQP2)-bearing vesicles to, and their subsequent fusion with, the plasma membrane (AQP2 shuttle). The AQP2 shuttle is evoked neither by AVP-independent Ca²⁺ increases nor by AVP-induced Ca²⁺ increases. However, clamping cytosolic Ca²⁺ concentrations below resting levels at 25 nM inhibited exocytosis. Exocytosis was confined to a slow monophasic response, and readily releasable vesicles were missing. Analysis of endocytic capacitance steps revealed that cAMP does not decelerate the retrieval of AQP2 from the plasma membrane. Our data suggest that cAMP initiates an early step, namely the transport of AQP2-bearing vesicles towards the plasma membrane, and do not support a regulatory function for Ca²⁺ in the AQP2 shuttle.     

Analysis of Flagellar Phosphoproteins from Chlamydomonas reinhardtii

Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.Cilia and flagella, which are essentially identical, are among the most ancient cellular organelles, providing motility for primitive eukaryotic cells living in aqueous environments. The assembly and motility of flagella have been studied extensively with the unicellular biflagellate green alga Chlamydomonas reinhardtii. This alga uses flagella for motility and for cell-cell recognition during mating. In basal land plants, such as bryophytes and pteridophytes, the only flagellated cells are motile sperm cells, which require water to swim to the egg. With the evolution of pollen tubes in higher gymnosperms and angiosperms, these plant species lost the ability to assemble flagella (24, 42). Flagella of animals have acquired new functions in multicellular organizations during evolution (6). In mammals, cilia and flagella can be motile or immotile. Motile cilia can be found, for example, in airways (respiratory cilia), in the brain (ependymal cilia), or in the male reproductive system (sperm flagella). Defects in cilia in humans can cause severe diseases, such as polycystic kidney disease, retinal degeneration, hydrocephalus, or changes in the left-right symmetry of organs, collectively known as ciliopathies (20, 32).Although C. reinhardtii and mammals are separated by more than 10⁹ years of evolution, C. reinhardtii flagella are amazingly similar in structure and function to the 9+2-type axonemes of most motile mammalian flagella and cilia (42). They are composed of nine microtubular doublets surrounding two central microtubular singlets. The axoneme of motile flagella includes substructures such as dynein arms and radial spokes that generate and control axoneme bending (31). The flagellum also contains matrix proteins that are not tightly associated with the flagellar membrane or the axoneme. They serve diverse functions and can be involved in intraflagellar transport (IFT) (37).Proteome analyses of cilia, including, for example, a human cilium, a mouse photoreceptor sensory cilium, and the flagella of the green alga Chlamydomonas reinhardtii, have unraveled hundreds of so far unknown proteins of this organelle (18, 29, 33) and have paved the way to further study the functions of these proteins. Several kinases and phosphatases were found in these proteomes, suggesting that reversible protein phosphorylation plays an important role in signaling in this organelle. This is underlined by earlier studies showing that phosphorylation and dephosphorylation control flagellar motility (35), signaling (30), length, and assembly (37, 53) in C. reinhardtii. Some phosphoproteins known or assumed to be involved in these processes, such as outer dynein arm heavy chain alpha (13), inner dynein arm intermediate chain protein IC138 (7), and central pair kinesin KLP1 (61), were characterized, but the exact in vivo phosphorylation sites were not determined. From earlier studies, it is known that >80 protein spots, representing axonemal components, are labeled by ³²P by two-dimensional electrophoretic techniques (34), but many of them have not been identified so far. In the past years, the relevance of some of the flagellar kinases has been shown. For example, silencing of casein kinase 1 (CK1) disturbs flagellum formation, among several other effects (41). One of its targets is IC138 (54). Glycogen synthase kinase 3 was suggested to regulate the assembly and length of flagella (53). Also, in mammalian cilia, reversible protein phosphorylation plays an important role in ciliary beating. Second messengers such as cyclic AMP (cAMP) and cGMP, which activate special kinases, are known to be relevant there (39).An understanding of how reversible protein phosphorylation influences the function of cilia and their role in diseases will require increased information not only about the nature of the phosphoproteins but also on their in vivo phosphorylation sites. In order to gain insight into the phosphoproteome of a eukaryotic cilium, we used the green alga C. reinhardtii, whose entire genome has been sequenced, as a model (23). This organism has many advantages for biochemical and molecular genetic studies of the flagellum. Importantly, as mentioned before, its flagellar proteome is known (33), and in addition, the proteome of the centriole that anchors the flagella is also known (11, 12).For the identification of the targets of the kinases and phosphatases in the flagella, phosphoproteomics can be applied. However, phosphoproteome analysis has been and still is a challenging task (19, 36, 47). This is due to a few facts, as follows. (i) Phosphoproteins can have more than one phosphorylation site, and the phosphorylation status of these sites can fluctuate depending on the physiological conditions of the cell. (ii) Only a small portion of a given protein in the cell can be phosphorylated. (iii) Furthermore, phosphoproteins, especially those of signaling pathways, are often proteins found in low abundance. Therefore, it is necessary to enrich the phosphopeptides. Among different methods, immobilized metal affinity chromatography (IMAC) is frequently used for phosphopeptide enrichment. In C. reinhardtii, phosphopeptides from proteins of the cellular, thylakoid, and eyespot phosphoproteomes were identified by this way (49, 50, 51, 52). Thereby, it became obvious that biochemical enrichment of subcellular fractions as it was done with the eyespot apparatus results in an increase of phosphopeptide identification (52). In this study, we used IMAC and tandem mass spectrometry (MS/MS) along with the acquisition of data-dependent neutral loss (MS/MS/MS spectra) to identify phosphopeptides from isolated flagella of C. reinhardtii. In this way, we identified 32 flagellar phosphoproteins, including different functional categories, along with 126 in vivo phosphorylation sites. In many cases, a dynamic phosphorylation pattern within one peptide was observed.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.     

Biochemistry of methanol-dependent acetogenesis in Eubacterium callanderi KIST612

Methanol is the simplest of all alcohols, is universally distributed in anoxic sediments as a result of plant material decomposition and is constantly attracting attention as an interesting substrate for anaerobes like acetogens that can convert bio-renewable methanol into value-added chemicals. A major drawback in the development of environmentally friendly but economically attractive biotechnological processes is the present lack of information on biochemistry and bioenergetics during methanol conversion in these bacteria. The mesophilic acetogen Eubacterium callanderi KIST612 is naturally able to consume methanol and produce acetate as well as butyrate. To grasp the full potential of methanol-based production of chemicals, we analysed the genes and enzymes involved in methanol conversion to acetate and identified the redox carriers involved. We will display a complete model for methanol-derived acetogenesis and butyrogenesis in Eubacterium callanderi KIST612, tracing the electron transfer routes and shed light on the bioenergetics during the process.     

Complex patterns of geographic variation in heat tolerance and Hsp70 expression levels in the common frog Rana temporaria

1.

We tested for geographical variation in heat tolerance and Hsp70 expression levels of Rana temporaria tadpoles along a 1500 km long latitudinal gradient in Sweden.