Solution structure of the NEAT (NEAr Transporter) domain from IsdH/HarA: the human hemoglobin receptor in Staphylococcus aureus

During infections the pathogen Staphylococcus aureus procures the essential nutrient iron from its host using iron-regulated surface determinant (Isd) proteins, which scavenge heme bound iron from host hemoproteins. Four Isd proteins are displayed in the cell wall, where they function as receptors for host proteins and heme. Each of the receptors contains one or more copies of a recently discovered domain called NEAT (NEAr Transporter) that has been shown to mediate protein binding. Here we report the three-dimensional solution structure of the NEAT domain from the IsdH/HarA protein, which is the hemoglobin receptor in the Isd system. This is the first structure of a NEAT domain and reveals that they adopt a beta sandwich fold that consists of two five-stranded antiparallel beta sheets. Although unrelated at the primary sequence level, our results indicate that NEAT domains belong to the immunoglobulin superfamily. Binding studies indicate that two IsdH/HarA NEAT domains bind a single molecule of methemoglobin, while the distantly related NEAT domain from the S. aureus IsdC protein binds only heme. A comparison of their primary sequences in light of the new structure is used to predict the hemoglobin and heme binding surfaces on NEAT domains.     

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin beta1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin beta1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin beta1, and consequently reversed TIMP-1-mediated integrin beta1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.     

Polyglutamine repeats and β‐helix structure: Molecular dynamics study

Neurodegenerative diseases are often associated with the formation of highly insoluble aggregates. Despite the efforts devoted to the characterization of these aggregates, their structure remains elusive. Several neurodegenerative diseases are characterized by the expansion of CAG repeats, which code for Gln. Among the structural models proposed for the aggregates observed in polyQ-linked diseases, the nanotube beta-helix model proposed by Perutz and colleagues Proc Natl Acad Sci U S A 2002;99:5591-5595 has been influential. In the present study, the stability of this beta-helix model has been investigated by performing molecular dynamics simulations on polyQ fragments of different lengths. The results indicate that models shorter than two full beta-helix turns are unstable and collapse toward irregular structures. On the other hand, longer beta-helix models, containing more than 40 residues, achieve a dynamic regular structure. This finding is in line with the observed threshold of Gln repeats (approximately 40) correlated with the insurgence of the disease. Notably, the structure of the final state of the models longer than 40 residues strictly depends on their size. A compact stable ellipsoidal structure is formed by the model made of two full helical turns (41 residues), whereas water filled tubular structures emerge from simulation on longer polypeptides. These results have been interpreted taking into account the experimental data on polyQ aggregates. A structural interpretation of the literature data has been proposed by assuming that different beta-helical models are involved in the different stages of the aggregation process.     

Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology

Reduced glutathione (GSH) is critical for many cellular processes, and both its intracellular and extracellular concentrations are tightly regulated. Intracellular GSH levels are regulated by two main mechanisms: by adjusting the rates of synthesis and of export from cells. Some of the proteins responsible for GSH export from mammalian cells have recently been identified, and there is increasing evidence that these GSH exporters are multispecific and multifunctional, regulating a number of key biological processes. In particular, some of the multidrug resistance-associated proteins (Mrp/Abcc) appear to mediate GSH export and homeostasis. The Mrp proteins mediate not only GSH efflux, but they also export oxidized glutathione derivatives (e.g., glutathione disulfide (GSSG), S-nitrosoglutathione (GS-NO), and glutathione-metal complexes), as well as other glutathione S-conjugates. The ability to export both GSH and oxidized derivatives of GSH, endows these transporters with the capacity to directly regulate the cellular thiol-redox status, and therefore the ability to influence many key signaling and biochemical pathways. Among the many processes that are influenced by the GSH transporters are apoptosis, cell proliferation, and cell differentiation. This report summarizes the evidence that Mrps contribute to the regulation of cellular GSH levels and the thiol-redox state, and thus to the many biochemical processes that are influenced by this tripeptide.     

Inheritance and variation of erucic acid content in a transgenic rapeseed (Brassica napus L.) doubled haploid population

Erucic acid (22:1) is a valuable renewable resource for the oleochemical industry. Currently available high erucic acid rapeseed cultivars contain only about 50% erucic acid in the seed oil. A substantial increase of the erucic acid content of the rapeseed oil could increase market prospects. The transgenic line TNKAT, over expressing the rapeseed fatty acid elongase gene (fae1) and expressing the Ld-LPAAT gene from Limnanthes douglasii was crossed with the line 6575-1 HELP (high erucic and low polyunsaturated fatty acid). A from the F₁ plants produced population of 90 doubled haploid (DH) lines was tested in a greenhouse with three replicates. Parental lines TNKAT and 6575-1 HELP contained 46 and 50% erucic acid in the seed oil, respectively. In the DH population the erucic acid content ranged between 35 and 59%. The Ld-LPAAT + Bn-fae1.1 transgene showed a 1:1 segregation. The transgenic DH lines contained up to 8% trierucolyglycerol, but surprisingly had a by 2.3% lower erucic acid content compared to the non-transgenic segregants. Results indicated that the ectopically expressed fae1.1 gene may not be functional. The DH population also showed a large quantitative variation for PUFA content ranging from 6 to 28% (TNKAT: 21%, 6575-1 HELP: 8%). Regression analysis showed that in the DH population a 10% reduction in PUFA content led to a 4.2% increase in erucic acid content. Development of locus specific PCR primers for the two resident erucic acid genes fae1.1 (A-genome) and fae1.2 genes (C-genome) of rapeseed allowed sequencing of the respective alleles from TNKAT and 6575-1 HELP. Single nucleotide polymorphisms were only found for the fae1.1 gene. Use of allele specific fae1.1 PCR primers, however, did not reveal a significant effect of the fae1.1 allele from either parent on erucic acid content. The high erucic acid low polyunsaturated fatty acid DH lines and the fae1 locus specific primers developed in the present study should be useful in future studies aimed at increasing erucic acid content in rapeseed.     

Human faecal microbiota display variable patterns of glycerol metabolism

Significant amounts of glycerol reach the colon microbiota daily through the diet and/or by in situ microbial production or release from desquamated epithelial cells. Some gut microorganisms may anaerobically reduce glycerol to 1,3-propanediol (1,3-PDO), with 3-hydroxypropanal as an intermediate. Accumulation of the latter intermediate may result in the formation of reuterin, which is known for its biological activity (e.g. antimicrobial properties). To date, glycerol metabolism in mixed cultures from the human colon has received little attention. Using in vitro batch incubations of faeces from 10 human individuals, we demonstrated that glycerol addition (140 mM) significantly affects the metabolism and composition of the microbial community. About a third of the samples exhibited rapid glycerol conversion, yielding proportionally higher levels of acetate and 1,3-PDO. In contrast, a slower glycerol metabolism resulted in higher levels of propionate. Furthermore, rapid glycerol metabolism correlated with significant shifts in the Lactobacillus-Enterococcus community, which were not observed in slower glycerol-metabolizing samples. As the conversion of glycerol to 1,3-PDO is a highly reducing process, we infer that the glycerol metabolism may act as an effective hydrogen sink. Given the importance of hydrogen-consuming processes in the gut, this work suggests that glycerol may have potential as a tool for modulating fermentation kinetics and profiles in the gastrointestinal tract.     

Structure of surface layer homology (SLH) domains from Bacillus anthracis surface array protein

Surface (S)-layers, para-crystalline arrays of protein, are deposited in the envelope of most bacterial species. These surface organelles are retained in the bacterial envelope through the non-covalent association of proteins with cell wall carbohydrates. Bacillus anthracis, a Gram-positive pathogen, produces S-layers of the protein Sap, which uses three consecutive repeats of the surface-layer homology (SLH) domain to engage secondary cell wall polysaccharides (SCWP). Using x-ray crystallography, we reveal here the structure of these SLH domains, which assume the shape of a three-prong spindle. Each SLH domain contributes to a three-helical bundle at the spindle base, whereas another α-helix and its connecting loops generate the three prongs. The inter-prong grooves contain conserved cationic and anionic residues, which are necessary for SLH domains to bind the B. anthracis SCWP. Modeling experiments suggest that the SLH domains of other S-layer proteins also fold into three-prong spindles and capture bacterial envelope carbohydrates by a similar mechanism.     

Coral-rudist bioconstructions in the Upper Cretaceous Haidach section (Gosau Group; Northern Calcareous Alps, Austria)

Summary In the area of Haidach (Northern Calcareous Alps, Austria), coral-rudist mounds, rudist biostromes, and bioclastic limestones and marls constitute an Upper Cretaceous shelf succession approximately 100 meters thick. The succession is part of the mixed siliciclasticcarbonate Gosau Group that was deposited at the northern margin of the Austroalpine microplate. In its lower part, the carbonate succession at Haidach comprises two stratal packages that each consists, from bottom to top, of a coral-rudist mound capped by a rudist biostrome which, in turn, is overlain by bioclastic limestones and, locally, marls. The coral-rudist mounds consist mainly of floatstones. The coral assemblage is dominated by Fungiina, Astreoina, Heterocoeniina andAgathelia asperella (stylinina). From the rudists, elevators (Vaccinites spp., radiolitids) and recumbents (Plagioptychus) are present. Calcareous sponges, sclerosponges, and octocorals are subordinate. The elevator rudists commonly are small; they settled on branched corals, coral heads, on rudists, and on biolastic debris. The rudists, in turn, provided settlement sites for corals. Predominantly plocoid and thamnasteroid coral growth forms indicate soft substrata and high sedimentation rates. The mounds were episodically smothered by carbonate mud. Many corals and rudists are coated by thick and diverse encrustations that indicate high nutrient level and/or turbid waters. The coral-rudist mounds are capped byVaccinites biostromes up to 5 m thick. The establishment of these biostromes may result from unfavourable environmental conditions for corals, coupled with the potential of the elevator rudists for effective substrate colonization. TheVaccinites biostromes are locally topped by a thin radiolitid biostrome. The biostromes, in turn, are overlain by bioclastic limestones; these are arranged in stratal packages that were deposited from carbonate sand bodies. Approximately midsection, an interval of marls with abundantPhelopteria is present. These marls were deposited in a quiet lagoonal area where meadows of sea grass or algae, coupled with an elevated nutrient level, triggered the mass occurrence ofPhelopteria. The upper part of the Haidach section consists of stratal packages that each is composed of a rudist biostrome overlain by bioclastic wackestones to packstones with diverse smaller benthic foraminifera and calcareous green algae. The biostromes are either built by radiolitids,Vaccinites, andPleurocora, or consist exclusively of radiolitids (mainlyRadiolites). Both the biostromes and the bioclastic limestones were deposited in a low-energy lagoonal environment that was punctuated by high-energy events.In situ-rudist fabrics typically have a matrix of mudstone to rudistclastic wackestone; other biogens (incl. smaller benthic foraminifera) are absent or very rare. The matrix of rudist fabrics that indicate episodic destruction by high-energy events contain a fossil assemblage similar to the vertically associated bioclastic limestones. Substrata colonized by rudists thus were unfavourable at least for smaller benthic foraminifera. The described succession was deposited on a gently inclined shelf segment, where coral-rudist mounds and hippuritid biostromes were separated by a belt of bioclastic sand bodies from a lagoon with radiolitid biostromes. The mounds document that corals and Late Cretaceous elevator rudists may co-occur in close association. On the scale of the entire succession, however, mainly as a result of the wide ecologic range of the rudists relative to corals, the coral-dominated mounds and the rudist biostromes are vertically separated.