Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.     

Insulin regulates GLUT1-mediated glucose transport in MG-63 human osteosarcoma cells

Osteosarcoma is the most common type of malignant bone cancer, accounting for 35% of primary bone malignancies. Because cancer cells utilize glucose as their primary energy substrate, the expression and regulation of glucose transporters (GLUT) may be important in tumor development and progression. GLUT expression has not been studied previously in human osteosarcoma cell lines. Furthermore, although insulin and insulin-like growth factor (IGF-I) play an important role in cell proliferation and tumor progression, the role of these hormones on GLUT expression and glucose uptake, and their possible relation to osteosarcoma, have also not been studied. We determined the effect of insulin and IGF-I on GLUT expression and glucose transport in three well-characterized human osteosarcoma cell lines (MG-63, SaOs-2, and U2-Os) using immunocytochemical, RT-PCR and functional kinetic analyses. Furthermore we also studied GLUT isoform expression in osteosarcoma primary tumors and metastases by in situ hybridization and immunohistochemical analyses. RT-PCR and immunostaining show that GLUT1 is the main isoform expressed in the cell lines and tissues studied, respectively. Immunocytochemical analysis shows that although insulin does not affect levels of GLUT1 expression it does induce a translocation of the transporter to the plasma membrane. This translocation is associated with increased transport of glucose into the cell. GLUT1 is the main glucose transporter expressed in osteosarcoma, furthermore, this transporter is regulated by insulin in human MG-63 cells. One possible mechanism through which insulin is involved in cancer progression is by increasing the amount of glucose available to the cancer cell.     

Rotavirus Viroplasm Proteins Interact with the Cellular SUMOylation System: Implications for Viroplasm-Like Structure Formation

Posttranslational modification by SUMO provides functional flexibility to target proteins. Viruses interact extensively with the cellular SUMO modification system in order to improve their replication, and there are numerous examples of viral proteins that are SUMOylated. However, thus far the relevance of SUMOylation for rotavirus replication remains unexplored. In this study, we report that SUMOylation positively regulates rotavirus replication and viral protein production. We show that SUMO can be covalently conjugated to the viroplasm proteins VP1, VP2, NSP2, VP6, and NSP5. In addition, VP1, VP2, and NSP2 can also interact with SUMO in a noncovalent manner. We observed that an NSP5 SUMOylation mutant protein retains most of its activities, such as its interaction with VP1 and NSP2, the formation of viroplasm-like structures after the coexpression with NSP2, and the ability to complement in trans the lack of NSP5 in infected cells. However, this mutant is characterized by a high degree of phosphorylation and is impaired in the formation of viroplasm-like structures when coexpressed with VP2. These results reveal for the first time a positive role for SUMO modification in rotavirus replication, describe the SUMOylation of several viroplasm resident rotavirus proteins, and demonstrate a requirement for NSP5 SUMOylation in the production of viroplasm-like structures.     

Molecular Characterization of a Strain of Squash Leaf Curl China Virus from the Philippines

The complete nucleotide sequence of infectious cloned DNA components (A and B) of the causal agent of squash leaf curl disease in the Philippines was determined. DNA‐A and DNA‐B comprise 2739 and 2705 nucleotides, respectively; the common region is 174 bases in length. Five ORFs were found in DNA‐A and two in DNA‐B. Partial dimeric clones containing DNA‐A and DNA‐B, constructed in a binary vector and transformed into Agrobacterium tumefaciens, induced systemic infection in agro‐inoculated pumpkin plants (Cucurbita moschata). The total DNA‐A sequence was most closely related to that of Squash leaf curl China virus (SLCCNV) (88% identity), although the existence of B component of SLCCNV has not been reported. The deduced coat protein was like that of SLCCNV (98% amino acid sequence identity) and the Philippines virus has low sequence identity to Squash leaf curl virus (SLCV) and Squash mild leaf curl virus (SMLCV) (63 and 64% total nucleotide sequence identities, respectively). From these results, we propose that the Philippines virus be designated Squash leaf curl China virus‐[Philippines] (SLCCNV‐[PH]).     

Expression pattern of the expanded noggin gene family in the planarian Schmidtea mediterranea

Noggin genes are mainly known as inhibitors of the Bone Morphogenetic Protein (BMP) signalling pathway. Noggin genes play an important role in various developmental processes such as axis formation and neural differentiation. In vertebrates, inhibition of the BMP pathway is usually carried out together with other inhibitory molecules: chordin and follistatin. Recently, it has been shown in planarians that the BMP pathway has a conserved function in the maintenance and re-establishment of the dorsoventral axis during homeostasis and regeneration. In an attempt to further characterize the BMP pathway in this model we have undertaken an in silico search of noggin genes in the genome of Schmidtea mediterranea. In contrast to other systems in which between one and four noggin genes have been reported, ten genes containing a noggin domain are present in S. mediterranea. These genes have been classified into two groups: noggin genes (two genes) and noggin-like genes (eight genes). Noggin-like genes are characterized by the presence of an insertion of 50–60 amino acids in the middle of the noggin domain. Here, we report the characterization of this expanded family of noggin genes in planarians as well as their expression patterns in both intact and regenerating animals. In situ hybridizations show that planarian noggin genes are expressed in a variety of cell types located in different regions of the planarian body.     

Cyclin D1 genetic heterozygosity regulates colonic epithelial cell differentiation and tumor number in ApcMin mice

Constitutive β-catenin/Tcf activity, the primary transforming events in colorectal carcinoma, occurs through induction of the Wnt pathway or APC gene mutations that cause familial adenomatous polyposis. Mice carrying Apc mutations in their germ line (Apc^Min) develop intestinal adenomas. Here, the crossing of Apc^Min with cyclin D1^−/− mice reduced the intestinal tumor number in animals genetically heterozygous or nullizygous for cyclin D1. Decreased tumor number in the duodenum, intestines, and colons of Apc^Min/cyclin D1^+/− mice correlated with reduced cellular proliferation and increased differentiation. Cyclin D1 deficiency reduced DNA synthesis and induced differentiation of colonic epithelial cells harboring mutant APC but not wild-type APC cells in vivo. In previous studies, the complete loss of cyclin D1 through homozygous genetic deletion conveyed breast tumor resistance. The protection of mice, genetically predisposed to intestinal tumorigenesis, through cyclin D1 heterozygosity suggests that modalities that reduce cyclin D1 abundance could provide chemoprotection.     

Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70°C) and thermostability (with a T₅₀ in the range 66 to 71°C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k_cat/K_m ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter⁻¹. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.The basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) produces astaxanthin (3-3′-dihydroxy-β,β-carotene-4,4 dione [17, 25]). Different industries have displayed great interest in this carotenoid pigment due to its attractive red-orange color and antioxidant properties, which has intensified the molecular and genetic study of this yeast. As a result, several genes involved in the astaxanthin biosynthetic pathway have been cloned and/or characterized, as well as some other genes such as those encoding actin (60), glyceraldehyde-3-phosphate dehydrogenase (56), endo-β-1,3-glucanase, and aspartic protease (4). In terms of the use of carbon sources, a β-amylase (9), and an α-glucosidase (33) with glucosyltransferase activity (12), as well as a yeast cell-associated invertase (41), have also been reported.Invertases or β-fructofuranosidases (EC 3.2.1.26) catalyze the release of β-fructose from the nonreducing termini of various β-d-fructofuranoside substrates. Yeast β-fructofuranosidases have been widely studied, including that of Saccharomyces cerevisiae (11, 14, 45, 46), Schizosaccharomyces pombe (36), Pichia anomala (40, 49), Candida utilis (5, 8), or Schwanniomyces occidentalis (2). They generally exhibit strong similarities where sequences are available, and they have been classified within family 32 of the glycosyl-hydrolases (GH) on the basis of their amino acid sequences. The catalytic mechanism proposed for the S. cerevisiae enzyme implies that an aspartate close to the N terminus (Asp-23) acts as a nucleophile, and a glutamate (Glu-204) acts as the acid/base catalyst (46). In addition, the three-dimensional structures of some enzymes in this family have been resolved, such as that of an exoinulinase from Aspergillus niger (var. awamori; 37) and the invertase from Arabidopsis thaliana (55).As well as hydrolyzing sucrose, β-fructofuranosidases from microorganisms may also catalyze the synthesis of short-chain fructooligosaccharides (FOS), in which one to three fructosyl moieties are linked to the sucrose skeleton by different glycosidic bonds depending on the source of the enzyme (3, 52). FOS are one of the most promising ingredients for functional foods since they act as prebiotics (44), and they exert a beneficial effect on human health, participating in the prevention of cardiovascular diseases, colon cancer, or osteoporosis (28). Currently, Aspergillus fructosyltransferase is the main industrial producer of FOS (15, 52), producing a mixture of FOS with an inulin-type structure, containing β-(2→1)-linked fructose-oligomers (¹F-FOS: 1-kestose, nystose, or ¹F-fructofuranosylnystose). However, there is certain interest in the development of novel molecules that may have better prebiotic and physiological properties. In this context, β-(2→6)-linked FOS, where this link exits between two fructose units (⁶F-FOS: 6-kestose) or between fructose and the glucosyl moiety (⁶G-FOS: neokestose, neonystose, and neofructofuranosylnystose), may have enhanced prebiotic properties compared to commercial FOS (29, 34, 54). The enzymatic synthesis of 6-kestose and other related β-(2→6)-linked fructosyl oligomers has already been reported in yeasts such as S. cerevisiae (11) or Schwanniomyces occidentalis (2) and in fungi such as Thermoascus aurantiacus (26) or Sporotrichum thermophile (27). However, the production of FOS included in the ⁶G-FOS series has not been widely reported in microorganisms, probably because they are not generally produced (2, 15) or because they represent only a minor biosynthetic product (e.g., with baker''s yeast invertase) (11). Most research into neo-FOS production has been carried out with Penicillium citrinum cells (19, 31, 32, 39). In this context, neokestose is the main transglycosylation product accumulated by whole X. dendrorhous cells from sucrose (30), although the enzyme responsible for this reaction remained uncharacterized.Here, we describe the molecular, phylogenetic, and biochemical characterization of an extracellular β-fructofuranosidase from X. dendrorhous. Kinetic studies of its hydrolytic activity were performed using different substrates, and we investigated its fructosyltransferase capacity. The functionality of the gene analyzed was verified through its heterologous expression, and a structural model of this enzyme based on the homologous invertase from A. thaliana has also been obtained.     

Structural and Molecular Basis of the Peroxynitrite-mediated Nitration and Inactivation of Trypanosoma cruzi Iron-Superoxide Dismutases (Fe-SODs) A and B: DISPARATE SUSCEPTIBILITIES DUE TO THE REPAIR OF TYR35 RADICAL BY CYS83 IN Fe-SODB THROUGH INTRAMOLECULAR ELECTRON TRANSFER*

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10⁴ m⁻¹ s⁻¹ and 4.3 ± 0.4 × 10⁴ m⁻¹ s⁻¹ at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr³⁵. Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys⁸³ mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys⁸³ present in Fe-SODB acts as an electron donor that repairs Tyr³⁵ radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.