Cutting off functional loops from homodimeric enzyme superoxide dismutase 1 (SOD1) leaves monomeric β-barrels

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.     

Metabolic flux analysis gives an insight on verapamil induced changes in central metabolism of HL-1 cells

Verapamil has been shown to inhibit glucose transport in several cell types. However, the consequences of this inhibition on central metabolism are not well known. In this study we focused on verapamil induced changes in metabolic fluxes in a murine atrial cell line (HL-1 cells). These cells were adapted to serum free conditions and incubated with 4 μM verapamil and [U-¹³C₅] glutamine. Specific extracellular metabolite uptake/production rates together with mass isotopomer fractions in alanine and glutamate were implemented into a metabolic network model to calculate metabolic flux distributions in the central metabolism. Verapamil decreased specific glucose consumption rate and glycolytic activity by 60%. Although the HL-1 cells show Warburg effect with high lactate production, verapamil treated cells completely stopped lactate production after 24 h while maintaining growth comparable to the untreated cells. Calculated fluxes in TCA cycle reactions as well as NADH/FADH₂ production rates were similar in both treated and untreated cells. This was confirmed by measurement of cell respiration. Reduction of lactate production seems to be the consequence of decreased glucose uptake due to verapamil. In case of tumors, this may have two fold effects; firstly depriving cancer cells of substrate for anaerobic glycolysis on which their growth is dependent; secondly changing pH of the tumor environment, as lactate secretion keeps the pH acidic and facilitates tumor growth. The results shown in this study may partly explain recent observations in which verapamil has been proposed to be a potential anticancer agent. Moreover, in biotechnological production using cell lines, verapamil may be used to reduce glucose uptake and lactate secretion thereby increasing protein production without introduction of genetic modifications and application of more complicated fed-batch processes.     

Cytokinin Profiles in the Conifer Tree <Emphasis Type="Italic">Abies nordmanniana</Emphasis>: Whole-Plant Relations in Year-Round Perspective

Conifer trees are routinely manipulated hormonally to increase flowering, branching, or adjust crown shape for production purposes. This survey of internal cytokinin levels provides a background for such treatments in Abies nordmanniana, a tree of great economic interest. Reference points in the crown and root system were sampled destructively in 4- and 6-year-old trees and analyzed for a range of cytokinins by LC-MS/MS. No seasonal patterns were detected in the root samples, and a major portion of cytokinin was in conjugated forms. Dramatic and consistent seasonal changes occurred in the crown, at levels 17–65 times higher than in the root. Predominant among crown cytokinins was ZR, except in the needles where IPR was also prominent. Within the crown, cytokinin profiles in different organs differed consistently. The leader bud showed a pronounced mid-June minimum, and a maximum later in summer. Subapical buds showed the same June minimum but peaked in mid autumn at a much lower level. Maxima in these buds were preceded by peaks in the subapical stem. Parallel patterns were observed in homologous tissues on branches.This pattern is consistent with two surges beginning in the uppermost stem tissues leading to subsequent accumulation or stimulated production within the buds. Strong differential hormonal profiles between adjacent buds with different fates agree with recent evidence of localized cytokinin production. The data suggest a reduced role of root-derived cytokinins in crown development. Practical cytokinin treatments for crown-shape regulation require close attention to dosage as well as precise timing and positioning.     

Identification of clinically relevant protein targets in prostate cancer with 2D-DIGE coupled mass spectrometry and systems biology network platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies.     

Prospects of yeast systems biology for human health: integrating lipid, protein and energy metabolism

The yeast Saccharomyces cerevisiae is a widely used model organism for studying cell biology, metabolism, cell cycle and signal transduction. Many regulatory pathways are conserved between this yeast and humans, and it is therefore possible to study pathways that are involved in disease development in a model organism that is easy to manipulate and that allows for detailed molecular studies. Here, we briefly review pathways involved in lipid metabolism and its regulation, the regulatory network of general metabolic regulator Snf1 (and its human homologue AMPK) and the proteostasis network with its link to stress and cell death. All the mentioned pathways can be used as model systems for the study of homologous pathways in human cells and a failure in these pathways is directly linked to several human diseases such as the metabolic syndrome and neurodegeneration. We demonstrate how different yeast pathways are conserved in humans, and we discuss the possibilities of using the systems biology approach to study and compare the pathways of relevance with the objective to generate hypotheses and gain new insights.     

Are environmental factors responsible for geographic variation in the sex ratio of the Greenlandic seed‐bug Nysius groenlandicus?

Until recently nothing indicated an unequal sex ratio in the widespread Greenland seed‐bug Nysius groenlandicus (Zetterstedt) (Heteroptera: Lygaeidae). However, recently populations more or less devoid of males were discovered in high arctic Northeast Greenland. This initiated an inspection of the entire material of the species collected in Greenland and now preserved at the Zoological Museum, University of Copenhagen. It was found that the sex ratio varied significantly among different locations. In most cases females were most abundant, but males were either scarce or absent only in samples from Northeast Greenland, indicating that here the species reproduces asexually. This paper demonstrates that the differing sex distributions can be explained by climatic factors (temperature, precipitation) and that the degree of continentality (distance from the open sea) promotes female‐biased sex ratios.     

Global patterns in the shape of species geographical ranges reveal range determinants

Aim Do species range shapes follow general patterns? If so, what mechanisms underlie those patterns? We show for 11,582 species from a variety of taxa across the world that most species have similar latitudinal and longitudinal ranges. We then seek to disentangle the roles of climate, extrinsic dispersal limitation (e.g. barriers) and intrinsic dispersal limitation (reflecting a species’ ability to disperse) as constraints of species range shape. We also assess the relationship between range size and shape. Location Global. Methods Range shape patterns were measured as the slope of the regression of latitudinal species ranges against longitudinal ranges for each taxon and continent, and as the coefficient of determination measuring the degree of scattering of species ranges from the 1:1 line (i.e. latitudinal range = longitudinal range). Two major competing hypotheses explaining species distributions (i.e. dispersal or climatic determinism) were explored. To this end, we compared the observed slopes and coefficients of determination with those predicted by a climatic null model that estimates the potential range shapes in the absence of dispersal limitation. The predictions compared were that species distribution shapes are determined purely by (1) intrinsic dispersal limitation, (2) extrinsic dispersal limitations such as topographic barriers, and (3) climate. Results  Using this methodology, we show for a wide variety of taxa across the globe that species generally have very similar latitudinal and longitudinal ranges. However, neither neutral models assuming random but spatially constrained dispersal, nor models assuming climatic control of species distributions describe range shapes adequately. The empirical relationship between the latitudinal and longitudinal ranges of species falls between the predictions of these competing models. Main conclusions We propose that this pattern arises from the combined effect of macroclimate and intrinsic dispersal limitation, the latter being the major determinant among restricted‐range species. Hence, accurately projecting the impact of climate change onto species ranges will require a solid understanding of how climate and dispersal jointly control species ranges.     

Complement resistance of Borrelia burgdorferi correlates with the expression of BbCRASP-1, a novel linear plasmid-encoded surface protein that interacts with human factor H and FHL-1 and is unrelated to Erp proteins

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.     

Lipid rafts in cytokine signaling

Lipid rafts are established as critical structures for a variety of cellular processes, including immune cell activation. Beyond their importance for initial immune cell activation at the immunological synapse, lipid rafts are now also being recognized as important sites for cytokine and growth factor signal transduction, both in immune cells as part of secondary regulatory processes, and in non-immune cells. This review summarizes current knowledge regarding the roles of rafts in cytokine signaling and emphasizes the need for measures to better standardize the study of rafts.     

HER-2/neu-mediated down-regulation of biglycan associated with altered growth properties

The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu(-) cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu(-) fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu(+) cells by transforming growth factor-β(1) nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by ～5000-fold increase in Bgn-mRNA expression in HER-2/neu(+) cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade.