Structural Insights into Cargo Recognition by the Yeast PTS1 Receptor

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a K_D of 0.19 μm. To analyze the structural basis for receptor-cargo recognition, the PTS1 and adjacent amino acids of Pcs60p were systematically scanned for Pex5p binding by an in vitro site-directed photo-cross-linking approach. The cross-linked binding regions of the receptor were subsequently identified by high resolution mass spectrometry. Most cross-links were found with TPR6, TPR7, as well as the 7C-loop of Pex5p. Surface plasmon resonance analysis revealed a bivalent interaction mode for Pex5p and Pcs60p. Interestingly, Pcs60p lacking its C-terminal tripeptide sequence was efficiently cross-linked to the same regions of Pex5p. The K_D value of the interaction of truncated Pcs60p and Pex5p was in the range of 7.7 μm. Isothermal titration calorimetry and surface plasmon resonance measurements revealed a monovalent binding mode for the interaction of Pex5p and Pcs60p lacking the PTS1. Our data indicate that Pcs60p contains a second contact site for its receptor Pex5p, beyond the C-terminal tripeptide. The physiological relevance of the ancillary binding region was supported by in vivo import studies. The bivalent binding mode might be explained by a two-step concept as follows: first, cargo recognition and initial tethering by the PTS1-receptor Pex5p; second, lock-in of receptor and cargo.     

Glycan Microheterogeneity at the PGT135 Antibody Recognition Site on HIV-1 gp120 Reveals a Molecular Mechanism for Neutralization Resistance

Broadly neutralizing antibodies have been isolated that bind the glycan shield of the HIV-1 envelope spike. One such antibody, PGT135, contacts the intrinsic mannose patch of gp120 at the Asn332, Asn392, and Asn386 glycosylation sites. Here, site-specific glycosylation analysis of recombinant gp120 revealed glycan microheterogeneity sufficient to explain the existence of a minor population of virions resistant to PGT135 neutralization. Target microheterogeneity and antibody glycan specificity are therefore important parameters in HIV-1 vaccine design.     

Morphogenesis of Pestiviruses: New Insights from Ultrastructural Studies of Strain Giraffe-1

Knowledge on the morphogenesis of pestiviruses is limited due to low virus production in infected cells. In order to localize virion morphogenesis and replication sites of pestiviruses and to examine intracellular virion transport, a cell culture model was established to facilitate ultrastructural studies. Based on results of virus growth kinetic analysis and quantification of viral RNA, pestivirus strain Giraffe-1 turned out to be a suitable candidate for studies on virion generation and export from culture cells. Using conventional transmission electron microscopy and single-tilt electron tomography, we found virions located predominately in the lumen of the endoplasmic reticulum (ER) in infected cells and were able to depict the budding process of virions at ER membranes. Colocalization of the viral core protein and the envelope glycoprotein E2 with the ER marker protein disulfide isomerase (PDI) was demonstrated by immunogold labeling of cryosections. Moreover, pestivirions could be shown in transport vesicles and the Golgi complex and during exocytosis. Interestingly, viral capsid protein and double-stranded RNA (dsRNA) were detected in multivesicular bodies (MVBs), which implies that the endosomal compartment plays a role in pestiviral replication. Significant cellular membrane alterations such as those described for members of the Flavivirus and Hepacivirus genera were not found. Based on the gained morphological data, we present a consistent model of pestivirus morphogenesis.     

Overlapping and Distinct Roles of Aspergillus fumigatus UDP-glucose 4-Epimerases in Galactose Metabolism and the Synthesis of Galactose-containing Cell Wall Polysaccharides

The cell wall of Aspergillus fumigatus contains two galactose-containing polysaccharides, galactomannan and galactosaminogalactan, whose biosynthetic pathways are not well understood. The A. fumigatus genome contains three genes encoding putative UDP-glucose 4-epimerases, uge3, uge4, and uge5. We undertook this study to elucidate the function of these epimerases. We found that uge4 is minimally expressed and is not required for the synthesis of galactose-containing exopolysaccharides or galactose metabolism. Uge5 is the dominant UDP-glucose 4-epimerase in A. fumigatus and is essential for normal growth in galactose-based medium. Uge5 is required for synthesis of the galactofuranose (Galf) component of galactomannan and contributes galactose to the synthesis of galactosaminogalactan. Uge3 can mediate production of both UDP-galactose and UDP-N-acetylgalactosamine (GalNAc) and is required for the production of galactosaminogalactan but not galactomannan. In the absence of Uge5, Uge3 activity is sufficient for growth on galactose and the synthesis of galactosaminogalactan containing lower levels of galactose but not the synthesis of Galf. A double deletion of uge5 and uge3 blocked growth on galactose and synthesis of both Galf and galactosaminogalactan. This study is the first survey of glucose epimerases in A. fumigatus and contributes to our understanding of the role of these enzymes in metabolism and cell wall synthesis.     

Two Fatty Acid Desaturases,STEAROYL-ACYL CARRIER PROTEIN Δ9-DESATURASE6 and FATTY ACID DESATURASE3, Are Involved in Drought and Hypoxia Stress Signaling in Arabidopsis Crown Galls

Agrobacterium tumefaciens-derived crown galls of Arabidopsis (Arabidopsis thaliana) contain elevated levels of unsaturated fatty acids and strongly express two fatty acid desaturase genes, ω3 FATTY ACID DESATURASE3 (FAD3) and STEAROYL-ACYL CARRIER PROTEIN Δ⁹-DESATURASE6 (SAD6). The fad3-2 mutant with impaired α-linolenic acid synthesis developed significantly smaller crown galls under normal, but not under high, relative humidity. This strongly suggests that FAD3 plays a role in increasing drought stress tolerance of crown galls. SAD6 is a member of the SAD family of as yet unknown function. Expression of the SAD6 gene is limited to hypoxia, a physiological condition found in crown galls. As no sad6 mutant exists and to link the function of SAD6 with fatty acid desaturation in crown galls, the lipid pattern was analyzed of plants with constitutive SAD6 overexpression (SAD6-OE). SAD6-OE plants contained lower stearic acid and higher oleic acid levels, which upon reduction of SAD6 overexpression by RNA interference (SAD6-OE-RNAi) regained wild-type-like levels. The development of crown galls was not affected either in SAD6-OE or SAD6-OE-RNAi or by RNA interference in crown galls. Since biochemical analysis of SAD6 in yeast (Saccharomyces cerevisiae) and Escherichia coli failed, SAD6 was ectopically expressed in the background of the well-known suppressor of salicylic acid-insensitive2 (ssi2-2) mutant to confirm the desaturase function of SAD6. All known ssi2-2 phenotypes were rescued, including the high stearic acid level. Thus, our findings suggest that SAD6 functions as a Δ⁹-desaturase, and together with FAD3 it increases the levels of unsaturated fatty acids in crown galls under hypoxia and drought stress conditions.Plant tumors, also referred to as crown galls, develop upon infection of susceptible plants with Agrobacterium tumefaciens. A DNA fragment, the transfer DNA (T-DNA) of the tumor-inducing plasmid of virulent A. tumefaciens strains, is randomly integrated into the genome of a host plant (Thomashow et al., 1980; Kim et al., 2007; Pitzschke and Hirt 2010). Expression of the T-DNA-encoded oncogenes drives increased production of auxin and cytokinin, thereby promoting cell proliferation. Plant tumor growth causes disruption of the epidermal cell layer that is covered by a cuticle and thus is permanently endangered to lose water (Schurr et al., 1996). In order to prevent desiccation and wilting, rescue processes appear to be constitutively activated (Veselov et al., 2003). Thereby, ethylene and abscisic acid trigger the expression of drought stress-responsive genes, the accumulation of osmoprotectants, and suberization of the surface cell layers (Efetova et al., 2007). In addition, cell membrane lipids are the major targets of environmental stresses, and tolerance to drought stress is dependent on high levels of polyunsaturated fatty acids (PUFAs) and the ability to maintain fatty acid (FA) desaturation activity (Berberich et al., 1998; Mikami and Murata, 2003; Torres-Franklin et al., 2009). In Arabidopsis (Arabidopsis thaliana) crown gall tumors, 27% of the genes involved in lipid metabolism are differentially regulated (Deeken et al., 2006). Under drought stress, Arabidopsis increases the ratio of digalactosyl diglyceride (DGDG) to monogalactosyl diglyceride (MGDG) and FA unsaturation (Gigon et al., 2004). An increase in α-linolenic acid levels (18:3, where x:y denotes an FA with x carbons and y double bonds) by overexpression of the ω3 fatty acid desaturases FAD3 and FAD7 has been shown to enhance tolerance to drought stress in Nicotiana tabacum cells (Zhang et al., 2005), whereas nontolerant plants decline their fraction of 18:3 (Monteiro de Paula et al., 1993; Dakhma et al., 1995; Upchurch, 2008).Developing crown gall tumors also face permanent oxidative stress. Reactive oxygen species (ROS) are produced in tumors, and glutathione S-transferases and peroxidases are strongly up-regulated (Jia et al., 1996; Lee et al., 2009). Due to a reduced respiratory and photosynthetic capacity in crown gall tumors, ATP production is predominantly derived from glycolysis and alcoholic fermentation (Deeken et al., 2006). In other words, the hypoxia physiology of the Arabidopsis tumor is governed by heterotrophic metabolism (Deeken et al., 2006). Since dioxygen is a cofactor of desaturases, its depletion limits the de novo synthesis of unsaturated FAs and thus membrane lipids (Brown and Beevers, 1987). In addition, hypoxia appears to be associated with ROS production, peroxidation of PUFAs, and finally, loss of membrane integrity (Blokhina et al., 2003; Upchurch, 2008).The biosynthesis of PUFAs is initiated by introduction of the first double bond into stearic acid (18:0) by STEAROYL-ACYL CARRIER PROTEIN Δ⁹-DESATURASE (SAD). SAD genes exhibit a tissue-specific expression profile, and the encoding enzymes regulate the pools of oleic acid (18:1), a monounsaturated fatty acid (MUFA; Shanklin and Somerville 1991; Thompson et al., 1991; Cahoon et al., 1996, 1998; Whittle et al., 2005). In Arabidopsis, five out of seven members of the SAD gene family (SAD1, SAD3, SAD4, SAD5, and SUPPRESSOR OF SALICYLIC ACID INSENSITIVE2 [SSI2]) are capable of desaturating 18:0 and contribute to the 18:1 pool (Kachroo et al., 2007). A mutation in the Arabidopsis ssi2 locus results in the accumulation of 18:0, whereas the 18:1 content is reduced. Furthermore, the salicylic acid-mediated defense signaling pathway is constitutively active, resulting in lesion formation and increased expression of the PATHOGENESIS-RELATED (PR) genes. The 18:1 MUFAs are incorporated into membrane lipids by two glycerolipid biosynthesis pathways. Phospholipids and galactolipids of photosynthetic membranes in plastids are synthesized by the prokaryotic pathway, while lipids of extraplastidic membranes are produced in the endoplasmic reticulum (ER) by the eukaryotic pathway (Ohlrogge and Browse, 1995). MUFAs are further desaturated to PUFAs by two sets of membrane-bound FADs. These enzymes are either located in plastids or the ER (Ohlrogge and Browse, 1995). In the ER, conversion of the unsaturated phospholipid FAs 18:1 to 18:2 and of 18:2 to 18:3 is carried out by the ω6 desaturase FAD2 and the ω3 desaturase FAD3, respectively (Browse et al., 1993; Okuley et al., 1994; Los and Murata, 1998).This study focuses on the role of desaturases in Arabidopsis crown galls in the context of drought and hypoxia stress. We document that crown galls produce increased levels of α-linolenic acid and strongly express the two FAD genes FAD3 and SAD6. In contrast to the well-known ω3 desaturase FAD3, the function of SAD6, a putative SAD, is unknown, and mutants for this gene are not available. However, the ability of SAD6 to replace the well-characterized SSI2 functionally in the ssi2-2 mutant suggests that SAD6 is a functional SAD. Overexpression of SAD6 decreased stearic acid and increased oleic acid levels. Down-regulation of SAD6 overexpression by RNA interference (RNAi) restored wild-type-like FA levels. The ability of SAD6 to influence the oleic acid levels together with the finding that SAD6 gene expression is restricted to hypoxia suggest that SAD6 catalyzes FA desaturation under hypoxic conditions. Unlike SAD6, the results obtained with the fad3-2 mutant impaired in α-linolenic acid biosynthesis indicate a role of FAD3 in increasing the drought stress tolerance of crown galls. Thus, both desaturases shape the pool of unsaturated FAs in drought stress- and oxidative stress-challenged Arabidopsis crown gall tumors.     

Thiouracil-Forming Bacteria Identified and Characterized upon Porcine In Vitro Digestion of Brassicaceae Feed

In recent years, the frequent detection of the banned thyreostat thiouracil (TU) in livestock urine has been related to endogenous TU formation following digestion of glucosinolate-rich Brassicaceae crops. Recently, it was demonstrated that, upon in vitro digestion of Brassicaceae, fecal bacteria induce TU detection in livestock (porcine livestock > bovines). Therefore, the present study was intended to isolate and identify bacteria involved in this intestinal TU formation upon Brassicaceae digestion and to gain more insight into the underlying mechanism in porcine livestock. Twenty porcine fecal inocula (gilts and multiparous sows) were assessed through static in vitro colonic-digestion simulations with rapeseed. After derivatization and extraction of the fecal suspensions, TU was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS²). On average, lower TU concentrations were observed in fecal colonic simulations in gilts (8.35 ng g⁻¹ rapeseed ± 3.42 [mean ± standard deviation]) than in multiparous sows (52.63 ng g⁻¹ ± 16.17), which correlates with maturation of the gut microbial population with age. Further exploration of the mechanism showed cell-dependent activity of the microbial conversion and sustained TU-forming activity after subjection of the fecal inoculum to moderate heat over a time span of up to 30 min. Finally, nine TU-producing bacterial species were successfully isolated and identified by a combination of biochemical and molecular techniques as Escherichia coli (n = 5), Lactobacillus reuteri (n = 2), Enterococcus faecium (n = 1), and Salmonella enterica subsp. arizonae (n = 1). This report demonstrates that endogenous formation of TU is Brassicaceae induced and occurs under colonic conditions most likely through myrosinase-like enzyme activity expressed by different common intestinal bacterial species.