Discrimination between lumenal and cytosolic sites of deglycosylation in endoplasmic reticulum-associated degradation of glycoproteins by using benzyl mannose in CHO cell lines

Recent studies demonstrated that deglycosylation step is a prerequisite for endoplasmic reticulum (ER)-associated degradation of misfolded glycoproteins. Here, we report the advantages of using benzyl mannose during pulse-chase experiments to study the subcellular location of the deglycosylation step in Chinese hamster ovary (CHO) cell lines. Benzyl mannose inhibited both the ER-to-cytosol transport of oligomannosides and the trimming of cytosolic-labeled oligomannosides by the cytosolic mannosidase in vivo. We pointed out the occurrence of two subcellular sites of deglycosylation. The first one is located in the ER lumen, and led to the formation of Man8GlcNAc2 (isomer B) in wild-type CHO cell line and Man4GlcNAc2 in Man-P-Dol-deficient cell line. The second one was revealed in CHO mutant cell lines for which a high rate of glycoprotein degradation was required. It occurred in the cytosol and led to the liberation of oligosaccharides species with one GlcNAc residue and with a pattern similar to the one bound onto glycoproteins. The cytosolic deglycosylation site was not specific for CHO mutant cell lines, since we demonstrated the occurrence of cytosolic pathway when the formation of truncated glycans was induced in wild-type cells.     

NMR of Redox Proteins of Plants, Yeasts and Photosynthetic Bacteria

NMR spectroscopy has evolved dramatically over the past 15 years, establishing a new, reliable methodology for studying biomacromolecules at atomic resolution. The three-dimensional structure and dynamics of a biomolecule or a biomolecular complex is only one of the main types of information available using NMR. The spectral assignment to the specific nuclei of a biostructure is a very precise reflection of their electronic environment. Any change in this environment due to a structural change, the binding of a ligand or the redox state of a redox cofactor, will be very sensitively reported by changes in the different NMR parameters. The capabilities of the NMR method are currently expanding dramatically and it is turning into a powerful means to study biosystems dynamically in exchange between different conformations, exchanging ligands, transient complexes, or the activation/inhibition of regulated enzymes. We review here several NMR studies that have appeared during the past 5 or 6 years in the field of redox proteins of plants, yeasts and photosynthetic bacteria. These new results illustrate the recent biomolecular NMR evolution and provide new physiological models for understanding the different types of electron transfer, including glutaredoxins, thioredoxins and their dependent enzymes, the ferredoxin-NADP oxidoreductase complex, flavodoxins, the plastocyanin-cytochrome f complex, and cytochromes c.     

IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology

All osteogenic cells (osteoclasts, osteoblasts) contribute individually to bone remodeling. Their cellular interactions control their cellular activities and the bone remodeling intensity. These interactions can be established either through a cell-cell contact, involving molecules of the integrin family, or by the release of many polypeptidic factors and/or their soluble receptor chains. These factors can act directly on osteogenic cells and their precursors to control differentiation, formation and functions (matrix formation, mineralization, resorption...). Here, we present the involvement of three groups of cytokines which seem to be of particular importance in bone physiology: interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) (TNF-alpha)/IL-1, and the more recently known triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL). The interactions between these three groups are presented within the framework of bone resorption pathophysiology such as tumor associated osteolysis. The central role of the OPG/RANK/RANKL triad is pointed out.     

Lateglacial and Holocene vegetation history in the mountain range of central Pyrenees

We present the pollen analysis of a new sedimentary sequence taken at La Pouretère ( 1720 m), in the mountain vegetation zone of the Marcadau valley (central Pyrenees). The Lateglacial and Holocene chronology is supported by six 14C-dating results. The complementary analysis of some vegetal macroremains, stomata, pollen-clusters and the use of pollen influx allows us to elucidate the dynamic of mountain species such as Pinus and specially Abies but also to infer the unusual part played by Betula at the beginning of the Postglacial period.     

Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.     

IL-1 receptor type 1 gene-deficient mice demonstrate an impaired host defense against pneumococcal meningitis

The fatality rate associated with Streptococcus pneumoniae meningitis remains high despite adequate antibiotic treatment. IL-1 is an important proinflammatory cytokine, which is up-regulated in brain tissue after the induction of meningitis. To determine the role of IL-1 in pneumococcal meningitis we induced meningitis by intranasal inoculation with 8 x 10(4) CFU of S. pneumoniae and 180 U of hyaluronidase in IL-1R type I gene-deficient (IL-1R(-/-)) mice and wild-type mice. Meningitis resulted in elevated IL-1alpha and IL-1beta mRNA and protein levels in the brain. The absence of an intact IL-1 signal was associated with a higher susceptibility to develop meningitis. Furthermore, the lack of IL-1 impaired bacterial clearance, as reflected by an increased number of CFU in cerebrospinal fluid of IL-1R(-/-) mice. The characteristic pleocytosis of meningitis was not significantly altered in IL-1R(-/-) mice, but meningitis was associated with lower brain levels of cytokines. The mortality was significantly higher and earlier in the course of the disease in IL-1R(-/-) mice. These results demonstrate that endogenous IL-1 is required for an adequate host defense in pneumococcal meningitis.     

Drosophila egghead encodes a beta 1,4-mannosyltransferase predicted to form the immediate precursor glycosphingolipid substrate for brainiac

The neurogenic Drosophila genes brainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis of egghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:beta Man beta 1,3-N-acetylglucosaminyltransferase (beta 3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAc beta 1-3Man beta 1-4Glc beta 1-1Cer. In the present study we demonstrate that egghead encodes a Golgi-located GDP-mannose:beta Glc beta 1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Man beta 1-4Glc beta 1-1Cer. Egghead is unique among eukaryotic glycosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. We propose that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids.     

Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase

Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems. Recently, we described the SUF system of Escherichia coli and Erwinia chrysanthemi as being important for Fe-S biogenesis under stressful conditions. The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet. Here we demonstrate that: (i) SufE and SufS are both cystosolic as all members of the SUF system; (ii) SufE is a homodimeric protein; (iii) SufE forms a complex with SufS as shown by the yeast two-hybrid system and by affinity chromatography; (iv) binding of SufE to SufS is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS. This is the first example of a two-component cysteine desulfurase enzyme.     

Myocardial ischemia and increased heart work modulate the phosphorylation state of eukaryotic elongation factor-2

Protein synthesis, in particular peptide chain elongation, is an energy-consuming biosynthetic process. AMP-activated protein kinase (AMPK) is a key regulatory enzyme involved in cellular energy homeostasis. Therefore, we tested the hypothesis that, as in liver, it could mediate the inhibition of protein synthesis by oxygen deprivation in heart by modulating the phosphorylation of eukaryotic elongation factor-2 (eEF2), which becomes inactive in its phosphorylated form. In anoxic cardiomyocytes, AMPK activation was associated with an inhibition of protein synthesis and an increase in phosphorylation of eEF2. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), did not mimic the effect of oxygen deprivation to inhibit protein synthesis in cardiomyocytes or lead to eEF2 phosphorylation in perfused hearts, suggesting that AMPK activation did not inhibit mTOR/p70 ribosomal protein S6 kinase (p70S6K) signaling. Human recombinant eEF2 kinase (eEF2K) was phosphorylated by AMPK in a time- and AMP-dependent fashion, and phosphorylation led to eEF2K activation, similar to that observed in extracts from ischemic hearts. In contrast, increasing the workload resulted in a dephosphorylation of eEF2, which was rapamycin-insensitive, thus excluding a role for mTOR in this effect. eEF2K activity was unchanged by increasing the workload, suggesting that the decrease in eEF2 phosphorylation could result from the activation of an eEF2 phosphatase.