Biosynthesisin vitro of neolactotetraosylceramide by a galactosyltransferase from mouse T-lymphoma: purification and kinetic studies; synthesis of neolacto and polylactosamine core

The galactosyltransferase, GalT-4, which catalyses the biosynthesisin vitro of neolactotetraosylceramide, nLcOse4Cer (Gal1-4GleNAc1-3Gal1-4Glc-Cer) from lactotriaosylceramide, LcOse3Cer (GlcNAc1-3Gal1-4Glc-Cer), and UDP-galactose has been purified 107 500-fold from a mineral oil induced mouse T-lyphoma P-1798, using affinity columns. The purified enzyme is partially stabilized in the presence of phospholipid liposomes. Two closely migrating protein bands of apparent molecular weights 56 kDa and 63 kDa were observed after sodium dodecyl sulfate polyacrylamide gel electrophoresis of highly purified mouse GalT-4. These two protein bands, when subjected to limited proteolysis, resulted in three peptides with identical mobilities indicating amino acid sequence identity between the proteins. Both protein bands from P-1798 gave a positive immunostain when tested with polyclonal antibody against bovine lactose synthetase (UDP-Gal:Glc 4-galactosyltransferase) following Western blot analysis on nitrocellulose paper. The enzyme has a pH optimum between 6.5 and 7.0 and like all other galactosyltransferases, GalT-4 has absolute requirements for divalent cation (Mn²⁺). TheK _m values for the substrate LcOse3Cer and donor UDP-galactose are 110 and 250 µm, respectively. Substrate competition studies with LcOse3Cer and either asialo-agalacto-1-acid glycoprotein orN-acetylglucosamine revealed that these reactions might be catalysed by the same protein. The only other glycolipid which showed acceptor activity toward the purified GalT-4 was iLcOse5Cer (GlcNAc1-1-3Gal1-4Lc3), the precursor for polylactosamine antigens. However, competition studies with these two active substrates using the most purified enzyme fraction, revealed that these two reactions might be catalysed by two different proteins since the experimental values were closer to the theoretical values calculated for two enzymes. Interestingly however, it seems that the GalT-4 from P-1798 has an absolute requirement for anN-acetylglucosamine residue in the substrate since the lyso-derivative (GlcNH₂1-3Gal1-4Glc-sphingosine) of the acceptor glycolipid LcOse3Cer is completely inactive as substrate while theK _m andV _max of the reacetylated substrate (GlcNac1-3Gal1-4Glc-acetylsphingosine) was comparable with LcOse3Cer. Autoradiography of the radioactive product formed by purified P-1798 GalT-4 confirmed the presence of nLcOse4Cer, as the product cochromatographed with authentic glycolipid. The monoclonal antibody IB-2, specific for nLcOse4Cer, also produced a positive immunostained band on TLC as well as giving a positive ELISA when tested with radioactive product obtained using a highly purified enzyme from mouse P-1798 T-lymphoma.Abbreviations EDTA ethylenediamine tetraacetate - ME -mercaptoethanol - PEG polyethylene glycol - PBS phosphate buffered saline - Suc sucrose - Mn²⁺ manganese - Gal galactose - GlcNAc N-acetylglucosamine - UDP-Gal Uridine diphosphate galactose - Ab antibody - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - ECB embryonic chicken brain - Cer ceramide - nLc4 or NlcOse4Cer Gal1-4GleNac1-3Gal1-4Glc-Cer, neoLactotetraosylceramide - Lc3 or LcOse3Cer GlcNac1-3Gal1-4Glc-Cer, lactotriaosylceramide - iLc5 iLcOse5Cer, GlcNAc1-3nLcOse4Cer - nLc6 nLcOse6Cer, Gal1-4iLcOse5Cer - SA^–Gal^–₁AGP asialo-agalacto1-acid glycoprotein - TLC thin layer chromatography     

Endoplasmic reticulum export sites and Golgi bodies behave as single mobile secretory units in plant cells

In contrast with animals, plant cells contain multiple mobile Golgi stacks distributed over the entire cytoplasm. However, the distribution and dynamics of protein export sites on the plant endoplasmic reticulum (ER) surface have yet to be characterized. A widely accepted model for ER-to-Golgi transport is based on the sequential action of COPII and COPI coat complexes. The COPII complex assembles by the ordered recruitment of cytosolic components on the ER membrane. Here, we have visualized two early components of the COPII machinery, the small GTPase Sar1p and its GTP exchanging factor Sec12p in live tobacco (Nicotiana tabacum) leaf epidermal cells. By in vivo confocal laser scanning microscopy and fluorescence recovery after photobleaching experiments, we show that Sar1p cycles on mobile punctate structures that track with the Golgi bodies in close proximity but contain regions that are physically separated from the Golgi bodies. By contrast, Sec12p is uniformly distributed along the ER network and does not accumulate in these structures, consistent with the fact that Sec12p does not become part of a COPII vesicle. We propose that punctate accumulation of Sar1p represents ER export sites (ERES). The sites may represent a combination of Sar1p-coated ER membranes, nascent COPII membranes, and COPII vectors in transit, which have yet to lose their coats. ERES can be induced by overproducing Golgi membrane proteins but not soluble bulk-flow cargos. Few punctate Sar1p loci were observed that are independent of Golgi bodies, and these may be nascent ERES. The vast majority of ERES form secretory units that move along the surface of the ER together with the Golgi bodies, but movement does not influence the rate of cargo transport between these two organelles. Moreover, we could demonstrate using the drug brefeldin A that formation of ERES is strictly dependent on a functional retrograde transport route from the Golgi apparatus.     

Hepcidin assay in serum by SELDI-TOF-MS and other approaches

Hepcidin, a liver peptide hormone, is the central regulator of iron homeostasis. Hepcidin synthesis is modulated by iron stores, so that iron repletion increases its levels to prevent pathological overload, while iron deficiency strongly inhibits hepcidin to allow an increase in iron absorption from duodenal cells. The emerging pivotal role of hepcidin in iron homeostasis, along with its important links with basic pathways like inflammation, makes the availability of an accurate hepcidin assay as a potentially powerful investigative tool to improve our understanding as well as our diagnostic/prognostic capabilities in many human diseases. There has been a great interest worldwide in developing a reliable and widely applicable assay of the hormone in biological fluids. Being optimal for low-molecular-weight biomarkers, SELDI-TOF-MS has emerged as a valid tool for hepcidin assay. Here we review recent results obtained with this technique, as well as with other Mass Spectrometry-based and immunological methods.     

Selfie and the City: A World-Wide,Large, and Ecologically Valid Database Reveals a Two-Pronged Side Bias in Na?ve Self-Portraits

Self-portraits are more likely to show the artist’s right than left cheek. This phenomenon may have a psychobiological basis: Self-portraitists often copy their subject from mirrors and, if they prefer to present their left cheek (more expressive due to right-lateralization of emotions) to the mirror, this would result in a right-cheek bias in the painting. We tested this hypothesis using SelfieCity (3200 selfies posted on Instagram from December 4 through 12, 2013 from New York, Sao Paulo, Berlin, Moskow, and Bangkok), which includes two selfie-taking styles: a “standard” (photograph of selfie-taker) and a “mirror” (photograph of mirror reflection of selfie-taker) style. We show that the first style reveals a left cheek bias, whereas the second reveals a right cheek bias. Thus side biases observed in a world-wide, large, and ecologically valid database of naïve self-portraits provide strong support for a role of psychobiological factors in the artistic composition of self-portraits.     

Extended cardiolipin anchorage to cytochrome c: a model for protein–mitochondrial membrane binding

Two models have been proposed to explain the interaction of cytochrome c with cardiolipin (CL) vesicles. In one case, an acyl chain of the phospholipid accommodates into a hydrophobic channel of the protein located close the Asn52 residue, whereas the alternative model considers the insertion of the acyl chain in the region of the Met80-containing loop. In an attempt to clarify which proposal offers a more appropriate explanation of cytochrome c–CL binding, we have undertaken a spectroscopic and kinetic study of the wild type and the Asn52Ile mutant of iso-1-cytochrome c from yeast to investigate the interaction of cytochrome c with CL vesicles, considered here a model for the CL-containing mitochondrial membrane. Replacement of Asn52, an invariant residue located in a small helix segment of the protein, may provide data useful to gain novel information on which region of cytochrome c is involved in the binding reaction with CL vesicles. In agreement with our recent results revealing that two distinct transitions take place in the cytochrome c–CL binding reaction, data obtained here support a model in which two (instead of one, as considered so far) adjacent acyl chains of the liposome are inserted, one at each of the hydrophobic sites, into the same cytochrome c molecule to form the cytochrome c–CL complex.     

The nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP‐glucose into the endoplasmic reticulum,are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana

Uridine 5′‐diphosphate (UDP)‐glucose is transported into the lumen of the endoplasmic reticulum (ER), and the Arabidopsis nucleotide sugar transporter AtUTr1 has been proposed to play a role in this process; however, different lines of evidence suggest that another transporter(s) may also be involved. Here we show that AtUTr3 is involved in the transport of UDP‐glucose and is located at the ER but also at the Golgi. Insertional mutants in AtUTr3 showed no obvious phenotype. Biochemical analysis in both AtUTr1 and AtUTr3 mutants indicates that uptake of UDP‐glucose into the ER is mostly driven by these two transporters. Interestingly, the expression of AtUTr3 is induced by stimuli that trigger the unfolded protein response (UPR), a phenomenon also observed for AtUTr1, suggesting that both AtUTr1 and AtUTr3 are involved in supplying UDP‐glucose into the ER lumen when misfolded proteins are accumulated. Disruption of both AtUTr1 and AtUTr3 causes lethality. Genetic analysis showed that the atutr1 atutr3 combination was not transmitted by pollen and was poorly transmitted by the ovules. Cell biology analysis indicates that knocking out both genes leads to abnormalities in both male and female germ line development. These results show that the nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP‐glucose into the ER, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.     

A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway

A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST‐GFP, to other organelles. GOLD36 showed partial distribution of ST‐GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein‐like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk‐flow marker to the cell surface was also compromised. Using a combination of classical mapping and next‐generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus ( At1g54030 ). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock‐out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post‐ER compartments and suggest that its export from the ER is a requirement to ensure steady‐state maintenance of the organelle’s organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER.     

The identification of a series of novel, soluble non-peptidic neuropeptide Y Y2 receptor antagonists

The identification and subsequent optimisation of a selective non-peptidic NPY Y2 antagonist series is described. This led to the development of amine 2, a selective, soluble NPY Y2 receptor antagonist with enhanced CNS exposure.