Characterization of rapeseed myrosinase-binding protein

Myrosinase-binding proteins (MBPs) were purified from seeds of Brassica napus L. (oilseed rape). The proteins were characterized with respect to amino-acid composition, peptide sequence and isoelectric points. Gel electrophoresis and Western blotting of protein extracts from mature seeds showed the existence of at least ten proteins reacting with a monoclonal anti-MBP antibody and ranging in molecular size from 110 to 30 kDa. Proteins other than MBP reacting with the anti-MBP antibody were assigned as myrosinase-binding protein-related proteins (MBPRPs). Two MBPRPs were purified by immunoaffinity chromatography and characterized with respect to partial amino-acid sequence. Sequence identities were found between MBP and MBPRP. Western blot analysis of protein extracts from different tissues of B. napus showed that MBPRP is present in the whole plant, whereas MBP mostly occurs in the mature seed. A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was used to investigate the occurrence of MBP and MBPRP in developing seeds of some species in the Brassicaceae family.Abbreviations FPLC fast protein liquid chromatography - MBP myrosinase-binding protein - MBPRP myrosinase-bindingprotein-telated protein - PBS phosphate-buffered saline     

Cholesterol sensing by CD81 is important for hepatitis C virus entry

CD81 plays a central role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the large extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol-sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus (HCV). Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81–cholesterol association but had disparate effects on HCV entry, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified a potential allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol-unbound) or closed (cholesterol-bound) conformation. The open mutant of CD81 exhibited reduced HCV receptor activity, whereas the closed mutant enhanced activity. These data are consistent with cholesterol sensing switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81–partner protein networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry, and CD81''s function as a molecular scaffold; these insights are relevant to CD81''s varied roles in both health and disease.     

An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.     

Enrichment of phosphoproteins and phosphopeptide derivatization identify universal stress proteins in elicitor-treated Arabidopsis

Protein phosphorylation is a key biological process that regulates reactions involved in plant-microbe interactions. The phosphorylated form of a protein often represents only a small fraction of the total population and can be problematic to analyze in a mass spectrometer. We demonstrate how a titanium dioxide (TiO(2)) resin can be employed for the enrichment of phosphoproteins, as well as a method to derivatize TiO(2)-purified phosphopeptides to facilitate determination of the exact site of phosphorylation. The use of these methods was exemplified by the identification of two plant proteins that were shown to be phosphorylated after the elicitation of Arabidopsis cells with Phytophthora infestans zoospores and xylanase. Both of the proteins that were identified, At5g54430.1 and At4g27320.1, were found to contain a universal stress protein domain with conserved residues for ATP binding.     

Triacylglycerols are synthesised and utilized by transacylation reactions in microsomal preparations of developing safflower (Carthamus tinctorius L.) seeds

Microsomal membrane preparations from the immature cotyledons of safflower (Carthamus tinctorius) catalysed the interconversion of the neutral lipids, mono-, di-, and triacylglycerol. Membranes were incubated with neutral lipid substrates, ¹⁴C-labelled either in the acyl or glycerol moiety, and the incorporation of radioactivity into other complex lipids determined. It was clear that diacylglycerol gave rise to triacylglycerol and monoacylglycerol as well as phosphatidylcholine. Radioactivity from added [¹⁴C] triacylglycerol was to a small extent transferred to diacylglycerol whereas added [¹⁴C] monoacylglycerol was rapidly converted to diacylglycerols and triacylglycerols. The formation of triacylglycerol from diacylglycerol occurred in the absence of acyl-CoA and hence did not involve diacylglycerol acyltransferase (DAGAT) activity. Monoacylglycerol was not esterified by direct acylation from acyl-CoA. We propose that these reactions were catalyzed by a diacylglycerol: diacylglycerol transacylase which yielded triacylglycerol and monoacylglycerol, the reaction being freely reversible. The specific activity of the transacylase was some 25% of the diacylglycerol acyltransferase activity and, hence, during the net accumulation of oil, substantial newly formed triacylglycerol equilibrated with the diacylglycerol pool. In its turn the diacylglycerol rapidly interconverted with phosphatidylcholine, the major complex lipid substrate for Δ12 desaturation. Hence, the oleate from triacylglycerols entering phosphatidylcholine via this route could be further desaturated to linoleate. A model is presented which reconciles these observations with our current understanding of fatty acid desaturation in phosphatidylcholine and oil assembly in oleaceous seeds. Received: 8 November 1996 / Accepted: 5 February 1997     

Herpes Zoster and Multiple Sclerosis

No significant difference was found between 50 consecutive patients with multiple sclerosis and matched controls in respect of previous infection with rubella or measles and chicken-pox, or of previous vaccination and immunizing injections. Significantly more patients had a past history of herpes zoster compared with the controls.     

Distribution of myrosinase in rapeseed tissues

Immunocytochemical studies on Brassica napus (rapeseed) tissues using a monoclonal antibody against myrosinase (thioglucoside glucohydrolase) showed that the enzyme was only present in a small number of cells. In the developing embryo, scattered myrosinase-containing cells were present in both cotyledons and axis. The enzyme accumulated in these cells during the later stages of seed development, approximately from day 20 until day 40 after pollination. Parallel staining with the immunocytochemical technique and a histochemical method identified these cells as myrosin cells. Myrosinase appeared to be located outside the myrosin grains, although the occasional association with the membrane of the grains also was noted. In leaves, petals, and siliques, scattered parenchyma cells were stained in the mesophyll as well as in the vascular tissue. In young leaves, guard cells also contained myrosinase. The enzyme was also present in xylem cells of the stem.     

Characterization of a Brassica napus myrosinase pseudogene: myrosinases are members of the BGA family of β-glycosidases

Myrosinase isoenzymes are known to be encoded by two different families of genes denoted MA and MB. Nucleotide sequence analysis of a Brassica napus genomic clone containing a gene for myrosinase revealed it to be a pseudogene of the MA family. The gene spans more than 5 kb and contains at least 12 exons. The exon sequence of the gene is highly similar to myrosinase cDNA sequences. However, the gene displays three potential or actual pseudogene characters. Southern blot analysis using probes from the 3 portions of the genomic and B. napus MA and MB cDNA clones showed that MA type myrosinases are encoded by approximately 4 genes, while MB type myrosinases are encoded by more than 10 genes in B. napus. Northern blots with mRNA from seeds and young leaves probed with the MA-and MB-specific probes showed that the MA and MB myrosinase gene families are differentially expressed. Myrosinases are highly similar to proteins of a -glycosidase enzyme family comprising both -glycosidases and phospho--glycosidases of as diverged species as archaebacteria, bacteria, mammals and plants. By homology to these -glycosidases, putative active site residues in myrosinase are discussed on the basis of the similarity between -glycosidases and cellulases.