Structural analysis of a peptide fragment of transmembrane transporter protein bilitranslocase

Using a combination of genomic and post-genomic approaches is rapidly altering the number of identified human influx carriers. A transmembrane protein bilitranslocase (TCDB 2.A.65) has long attracted attention because of its function as an organic anion carrier. It has also been identified as a potential membrane transporter for cellular uptake of several drugs and due to its implication in drug uptake, it is extremely important to advance the knowledge about its structure. However, at present, only the primary structure of bilitranslocase is known. In our work, transmembrane subunits of bilitranslocase were predicted by a previously developed chemometrics model and the stability of these polypeptide chains were studied by molecular dynamics (MD) simulation. Furthermore, sodium dodecyl sulfate (SDS) micelles were used as a model of cell membrane and herein we present a high-resolution 3D structure of an 18 amino acid residues long peptide corresponding to the third transmembrane part of bilitranslocase obtained by use of multidimensional NMR spectroscopy. It has been experimentally confirmed that one of the transmembrane segments of bilitranslocase has alpha helical structure with hydrophilic amino acid residues oriented towards one side, thus capable of forming a channel in the membrane.     

Secretion of Soluble Vascular Endothelial Growth Factor Receptor 1 (sVEGFR1/sFlt1) Requires Arf1, Arf6, and Rab11 GTPases

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35)S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35)S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.     

TNFα regulates the localization of CD40 in lipid rafts of glioma cells

Resistance of glioblastoma multiforme (GBM) to TNFα induced apoptosis is attributed to NFκB activation. As TNF-receptor family member CD40 regulates NFκB activation, we investigated the role of CD40 in NFκB activation in GBM. We observed elevated CD40 levels in human glioma samples as compared to the surrounding normal tissue. Treatment with TNFα elevated CD40 levels in glioma cells and inhibition of CD40 signaling failed to abrogate TNFα induced NFκΒ activity. While TNFα increased the interaction between TRAF2/6, IκBα, IKKα/β in the CD40 signalosome, the level of CD40 in the signalosome remained unaffected upon TNFα treatment. Interestingly, TNFα decreased the spatial localization of CD40 and increased TRAF2/6 co-localization with lipid raft marker Caveolin. As localization of CD40 signalosome in lipid raft is crucial for NFκB activation, TNFα mediated decreased clustering of CD40 in lipid rafts could have possibly contributed to its non-involvement in NFκB activation.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INCORPORATION OF N-GLYCOLYLHEXOSAMINES INTO MAMMALIAN GLYCANS BY FEEDING N-GLYCOLYLGALACTOSAMINE

The outermost positions of mammalian cell-surface glycans are predominantly occupied by the sialic acids N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). To date, hydroxylation of CMP-Neu5Ac resulting in the conversion into CMP-Neu5Gc is the only known enzymatic reaction in mammals to synthesize a monosaccharide carrying an N-glycolyl group. In our accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, jbc.M112.363549), we report a metabolic pathway for degradation of Neu5Gc, demonstrating that N-acetylhexosamine pathways are tolerant toward the N-glycolyl substituent of Neu5Gc breakdown products. In this study, we show that exogenously added N-glycolylgalactosamine (GalNGc) serves as a precursor for Neu5Gc de novo biosynthesis, potentially involving seven distinct mammalian enzymes. Following the GalNAc salvage pathway, UDP-GalNGc is epimerized to UDP-GlcNGc, which might compete with the endogenous UDP-GlcNAc for the sialic acid biosynthetic pathway. Using UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase-deficient cells, we confirm that conversion of GalNGc into Neu5Gc depends on this key enzyme of sialic acid biosynthesis. Furthermore, we demonstrate by mass spectrometry that the metabolic intermediates UDP-GalNGc and UDP-GlcNGc serve as substrates for assembly of most major classes of cellular glycans. We show for the first time incorporation of GalNGc and GlcNGc into chondroitin/dermatan sulfates and heparan sulfates, respectively. As demonstrated by structural analysis, N-glycolylated hexosamines were found in cellular gangliosides and incorporated into Chinese hamster ovary cell O-glycans. Remarkably, GalNAc derivatives altered the overall O-glycosylation pattern as indicated by the occurrence of novel O-glycan structures. This study demonstrates that mammalian N-acetylhexosamine pathways and glycan assembly are surprisingly tolerant toward the N-glycolyl substituent.     

Hydrogen sulfide inhibits high glucose-induced matrix protein synthesis by activating AMP-activated protein kinase in renal epithelial cells

Hydrogen sulfide, a signaling gas, affects several cell functions. We hypothesized that hydrogen sulfide modulates high glucose (30 mm) stimulation of matrix protein synthesis in glomerular epithelial cells. High glucose stimulation of global protein synthesis, cellular hypertrophy, and matrix laminin and type IV collagen content was inhibited by sodium hydrosulfide (NaHS), an H(2)S donor. High glucose activation of mammalian target of rapamycin (mTOR) complex 1 (mTORC1), shown by phosphorylation of p70S6 kinase and 4E-BP1, was inhibited by NaHS. High glucose stimulated mTORC1 to promote key events in the initiation and elongation phases of mRNA translation: binding of eIF4A to eIF4G, reduction in PDCD4 expression and inhibition of its binding to eIF4A, eEF2 kinase phosphorylation, and dephosphorylation of eEF2; these events were inhibited by NaHS. The role of AMP-activated protein kinase (AMPK), an inhibitor of protein synthesis, was examined. NaHS dose-dependently stimulated AMPK phosphorylation and restored AMPK phosphorylation reduced by high glucose. Compound C, an AMPK inhibitor, abolished NaHS modulation of high glucose effect on events in mRNA translation as well as global and matrix protein synthesis. NaHS induction of AMPK phosphorylation was inhibited by siRNA for calmodulin kinase kinase β, but not LKB1, upstream kinases for AMPK; STO-609, a calmodulin kinase kinase β inhibitor, had the same effect. Renal cortical content of cystathionine β-synthase and cystathionine γ-lyase, hydrogen sulfide-generating enzymes, was significantly reduced in mice with type 1 diabetes or type 2 diabetes, coinciding with renal hypertrophy and matrix accumulation. Hydrogen sulfide is a newly identified modulator of protein synthesis in the kidney, and reduction in its generation may contribute to kidney injury in diabetes.     

Estimating the global distribution of field size using crowdsourcing

There is an increasing evidence that smallholder farms contribute substantially to food production globally, yet spatially explicit data on agricultural field sizes are currently lacking. Automated field size delineation using remote sensing or the estimation of average farm size at subnational level using census data are two approaches that have been used. However, both have limitations, for example, automatic field size delineation using remote sensing has not yet been implemented at a global scale while the spatial resolution is very coarse when using census data. This paper demonstrates a unique approach to quantifying and mapping agricultural field size globally using crowdsourcing. A campaign was run in June 2017, where participants were asked to visually interpret very high resolution satellite imagery from Google Maps and Bing using the Geo‐Wiki application. During the campaign, participants collected field size data for 130 K unique locations around the globe. Using this sample, we have produced the most accurate global field size map to date and estimated the percentage of different field sizes, ranging from very small to very large, in agricultural areas at global, continental, and national levels. The results show that smallholder farms occupy up to 40% of agricultural areas globally, which means that, potentially, there are many more smallholder farms in comparison with the two different current global estimates of 12% and 24%. The global field size map and the crowdsourced data set are openly available and can be used for integrated assessment modeling, comparative studies of agricultural dynamics across different contexts, for training and validation of remote sensing field size delineation, and potential contributions to the Sustainable Development Goal of Ending hunger, achieve food security and improved nutrition and promote sustainable agriculture.     

Role of central nervous system and ovarian insulin receptor substrate 2 signaling in female reproductive function in the mouse

Insulin receptor signaling regulates female reproductive function acting in the central nervous system and ovary. Female mice that globally lack insulin receptor substrate (IRS) 2, which is a key mediator of insulin receptor action, are infertile with defects in hypothalamic and ovarian functions. To unravel the tissue-specific roles of IRS2, we examined reproductive function in female mice that lack Irs2 only in the neurons. Surprisingly, these animals had minimal defects in pituitary and ovarian hormone levels, ovarian anatomy and function, and breeding performance, which indicates that the central nervous system IRS2 is not an obligatory signaling component for the regulation of reproductive function. Therefore, we undertook a detailed analysis of ovarian function in a novel Irs2 global null mouse line. Comparative morphometric analysis showed reduced follicle size, increased numbers of atretic follicles, as well as impaired oocyte growth and antral cavity development in Irs2 null ovaries. Granulosa cell proliferation was also defective in the Irs2 null ovaries. Furthermore, the insulin- and eCG-stimulated phosphoinositide-3-OH kinase signaling events, which included phosphorylation of Akt/protein kinase B and glycogen synthase kinase 3-beta, were impaired, whereas mitogen-activated protein kinase signaling was preserved in Irs2 null ovaries. These abnormalities were associated with reduced expression of cyclin D2 and increased CDKN1B levels, which indicates dysregulation of key components of the cell cycle apparatus implicated in ovarian function. Our data suggest that ovarian rather than central nervous system IRS2 signaling is important in the regulation of female reproductive function.     

Synergistic effect of TRM2/RNC1 and EXO1 in DNA double-strand break repair in Saccharomyces cerevisiae

In our recently published study, we provided in vitro as well as in vivo data demonstrating the involvement of TRM2/RNC1 in homologous recombination based repair (HRR) of DNA double strand breaks (DSBs), in support of such claims reported earlier. To further validate its role in DNA DSB processing, our present study revealed that the trm2 single mutant displays higher sensitivity to persistent induction of specific DSBs at the MAT locus by HO-endonuclease with higher sterility rate among the survivors compared to wild type (wt) or exo1 single mutants. Intriguingly, both sensitivity and sterility rate increased dramatically in trm2exo1 double mutants lacking both endo-exonucleases with a progressively increased sterility rate in trm2exo1 double mutants with short-induction periods, reaching a very high level of sterility with persistent DSB inductions. Mutation analysis of the mating type (MAT) locus among the sterile survivors with persistent HO-induction in trm2 and exo1 single mutants as well as in trm2exo1 double mutants revealed a similar small insertions and deletions events, characteristic of non-homologous end joining (NHEJ) that might have occurred due to the lack of proper processing function in these mutants. In addition, trm2ku80 and trm2rad52 double mutants also displayed significantly higher sterility with persistent DSB induction compared to ku80 and rad52 single mutants, respectively, exhibiting a mutation spectra that shifted from base substitution (in ku80 and rad52 single mutants) to small insertions and deletions in the double mutants (in trm2ku80 and trm2rad52 mutants). These data indicate a defective processing in absence of TRM2, with a synergistic effect of TRM2, and EXO1 in such processing.     

Composition and distribution of benthic isopod (Crustacea,Malacostraca) families off the Victoria-Land Coast (Ross Sea,Antarctica)

The benthic fauna off the Victoria-Land-Coast, Ross Sea (Antarctica) was investigated during the 19th Italica expedition in February 2004. Samples were taken along a latitudinal transect from Cape Adare down to Terra Nova Bay at water depths ranging from 84 to 515 m. A Rauschert dredge was used at 18 stations to collect epi- and infaunal macrobenthos. 9,494 specimens of Isopoda were collected, representing 19 families. Desmosomatidae were the most abundant family (35,297 ind/1,000 m²), followed by Paramunnidae (23,973 ind/1,000 m²). Paramunnidae was the most frequent taxon and was collected at all stations, in contrast to the Desmosomatidae, which did not occur at any station off Cape Adare.