Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage.

The proteasome is able to create spliced Ags, in which two distant parts of a protein are excised and ligated together to form a novel peptide, for presentation by MHC class I molecules. These noncontiguous epitopes are generated via a transpeptidation reaction catalyzed by the proteasomal active sites. Transpeptidation reactions in the proteasome follow explicit rules and occur particularly efficiently when the C-terminal ligation partner contains a lysine or arginine residue at the site of ligation. Lysine contains two amino groups that theoretically may both participate in ligation reactions, implying that potentially not only peptide but also isopeptide linkages could be formed. Using nuclear magnetic resonance spectroscopy, we demonstrate in the present study that the proteasome can use the ε-amino group of an N-terminal lysine residue in transpeptidation reactions to create a novel type of posttranslationally modified epitopes. We show that the overall efficiency of ε ligation is only 10-fold lower as compared with α ligation, suggesting that the proteasome can produce sufficient isopeptide Ag to evoke a T cell response. Additionally, we show that isopeptides are more stable toward further proteasomal processing than are normal peptides, and we demonstrate that isopeptides can bind to HLA-A2.1 and HLA-A3 with high affinity. These properties likely increase the fraction of ε-ligated peptides presented on the cell surface for CD8(+) T cell surveillance. Finally, we show that isopeptide Ags are immunogenic in vivo. We postulate that ε ligation is a genuine posttranslational modification, suggesting that the proteasome can create a novel type of Ag that is likely to play a role in immunity.

Structural identification of ladderane and other membrane lipids of planctomycetes capable of anaerobic ammonium oxidation (anammox).

The membrane lipid composition of planctomycetes capable of the anaerobic oxidation of ammonium (anammox), i.e. Candidatus'Brocadia anammoxidans' and Candidatus'Kuenenia stuttgartiensis', was shown to be composed mainly of so-called ladderane lipids. These lipids are comprised of three to five linearly concatenated cyclobutane moieties with cis ring junctions, which occurred as fatty acids, fatty alcohols, alkyl glycerol monoethers, dialkyl glycerol diethers and mixed glycerol ether/esters. The highly strained ladderane moieties were thermally unstable, which resulted in breakdown during their analysis with GC. This was shown by isolation of a thermal product of these ladderanes and subsequent analysis with two-dimensional NMR techniques. Comprehensive MS and relative retention time data for all the encountered ladderane membrane lipids is reported, allowing the identification of ladderanes in other bacterial cultures and in the environment. The occurrence of ladderane lipids seems to be limited to the specific phylogenetic clade within the Planctomycetales able to perform anammox. This was consistent with their proposed biochemical function, namely as predominant membrane lipids of the so-called anammoxosome, the specific organelle where anammox catabolism takes place in the cell.

A mixed ladderane/n-alkyl glycerol diether membrane lipid in an anaerobic ammonium-oxidizing bacterium.

A novel glycerol diether containing ladderane and tetradecyl moieties has been identified in an anaerobic ammonium-oxidizing bacterium by GC/MS and high-field NMR spectroscopy.

C(25) highly branched isoprenoid alkenes from the marine benthic diatom Pleurosigma strigosum.

The hydrocarbon composition of the marine diatom Pleurosigma strigosum isolated from coastal Mediterranean sediments is described. A suite of five C(25) highly branched isoprenoid (HBI) alkenes with 2-5 double bonds were detected together with n-C(21:4) and n-C(21:5) alkenes and squalene. The analysis by (1)H and (13)C NMR spectroscopy of two isolated HBI alkenes allowed the structural identification of a novel C(25) HBI triene (2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadeca-5E,13-diene) and the first identification in diatom cells of 2,6,10,14-tetramethyl-7-(3-methylpent-4-enyl)-pentadec-5E-ene, an HBI previously detected in marine sediments and particulate matter. The other minor C(25) HBIs detected were a tetraene and a pentaene that have been previously identified in other diatoms from the genera Haslea and Rhizosolenia, and one other C(25) tetraene that could not be structurally identified. The structures of the HBI alkenes of P. strigosum were compared with those of C(25) homologues previously identified in three other Pleurosigma sp. (Pleurosigma intermedium, Pleurosigma planktonicum and Pleurosigma sp.). Unlike most structures previously reported, none of the HBI alkenes produced by P. strigosum showed an unsaturation at C7-C20, or E/Z isomerism of the trisubstituted double bond at C9-C10 (whenever present).

Water lubricates hydrogen-bonded molecular machines.

The mechanical behaviour of molecular machines differs greatly from that of their macroscopic counterparts. This applies particularly when considering concepts such as friction and lubrication, which are key to optimizing the operation of macroscopic machinery. Here, using time-resolved vibrational spectroscopy and NMR-lineshape analysis, we show that for molecular machinery consisting of hydrogen-bonded components the relative motion of the components is accelerated strongly by adding small amounts of water. The translation of a macrocycle along a thread and the rotation of a molecular wheel around an axle both accelerate significantly on the addition of water, whereas other protic liquids have much weaker or opposite effects. We tentatively assign the superior accelerating effect of water to its ability to form a three-dimensional hydrogen-bond network between the moving parts of the molecular machine. These results may indicate a more general phenomenon that helps explain the function of water as the 'lubricant of life'.

A reanalysis of phospholipid fatty acids as ecological biomarkers for methanotrophic bacteria.

Aerobic methane-oxidizing bacteria (MB) are the primary terrestrial sinks for the greenhouse gas methane. A distinct characteristic of MB is the presence of specific phospholipid ester-linked fatty acids (PLFA) in their membranes that differentiate them from each other and also from all other organisms. These distinct PLFA patterns facilitate microbial ecology studies. For example, the assimilation of C from methane into PLFA can be traced in environmental samples using stable isotope ((13)C) probing (SIP), which links the activity of MB to their community composition in situ. However, the phylogenetic resolution of this method is low because of a lack of PLFA profiles from cultured MB species. In this study, PLFA profiles of 22 alphaproteobacterial (type II) MB were analysed after growth on methane, methanol or both substrates together. Growth on different substrates did not affect the PLFA profiles of the investigated strains. A number of Methylocystis strains contained novel C18:2 fatty acids (omega 7c,12c and omega 6c,12c) that can be used as diagnostic biomarkers. The detection of these novel PLFA, combined with the analyses of multiple type II strains, increased the phylogenetic resolution of PLFA analysis substantially. Multivariate analysis of the expanded MB PLFA database identified species groups that closely reflected phylogenies based on 16S rRNA and pmoA gene sequences. The PLFA database therefore provides a robust framework for linking identity to activity in MB communities with a higher resolution than was previously possible.

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway.

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-(13)C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field (13)C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C. Brocadia fulgida synthesizes C(16:0) and isoC(16:0) fatty acids according to the known pathway of type II fatty acid biosynthesis. The (13)C-labelling pattern of the C(8) alkyl chain of the C(20) [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.

Membrane lipids of mesophilic anaerobic bacteria thriving in peats have typical archaeal traits.

The 16S ribosomal DNA based distinction between the bacterial and archaeal domains of life is strongly supported by the membrane lipid composition of the two domains; Bacteria generally contain dialkyl glycerol diester lipids, whereas Archaea produce isoprenoid dialkyl glycerol diether and membrane-spanning glycerol dialkyl glycerol tetraether (GDGT) lipids. Here we show that a new group of ecologically abundant membrane-spanning GDGT lipids, containing branched instead of isoprenoid carbon skeletons, are of a bacterial origin. This was revealed by examining the stereochemistry of the glycerol moieties of those branched tetraether membrane lipids, which was found to be the bacterial 1,2-di-O-alkyl-sn-glycerol stereoconfiguration and not the 2,3-di-O-alkyl-sn-glycerol stereoconfiguration as in archaeal membrane lipids. In addition, unequivocal evidence for the presence of cyclopentyl moieties in these bacterial membrane lipids was obtained by NMR. The biochemical traits of biosynthesis of tetraether membrane lipids and the formation of cyclopentyl moieties through internal cyclization, which were thought to be specific for the archaeal lineage of descent, thus also occur in the bacterial domain of life.

Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether membrane lipid of cosmopolitan pelagic crenarchaeota.

The basic structure and stereochemistry of the characteristic glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipid of cosmopolitan pelagic crenarchaeota has been identified by high field two-dimensional (2D)-NMR techniques. It contains one cyclohexane and four cyclopentane rings formed by internal cyclisation of the biphytanyl chains. Its structure is similar to that of GDGTs biosynthesized by (hyper)thermophilic crenarchaeota apart from the cyclohexane ring. These findings are consistent with the close phylogenetic relationship of (hyper)thermophilic and pelagic crenarchaeota based 16S rRNA. The latter group inherited the biosynthetic capabilities for a membrane composed of cyclopentane ring-containing GDGTs from the (hyper)thermophilic crenarchaeota. However, to cope with the much lower temperature of the ocean, a small but key step in their evolution was the adjustment of the membrane fluidity by making a kink in one of the bicyclic biphytanyl chains by the formation of a cyclohexane ring. This prevents the dense packing characteristic for the cyclopentane ring-containing GDGTs membrane lipids used by hyperthermophilic crenarchaeota to adjust their membrane fluidity to high temperatures.

Linearly concatenated cyclobutane lipids form a dense bacterial membrane.

Lipid membranes are essential to the functioning of cells, enabling the existence of concentration gradients of ions and metabolites. Microbial membrane lipids can contain three-, five-, six- and even seven-membered aliphatic rings, but four-membered aliphatic cyclobutane rings have never been observed. Here we report the discovery of cyclobutane rings in the dominant membrane lipids of two anaerobic ammonium-oxidizing (anammox) bacteria. These lipids contain up to five linearly fused cyclobutane moieties with cis ring junctions. Such 'ladderane' molecules are unprecedented in nature but are known as promising building blocks in optoelectronics. The ladderane lipids occur in the membrane of the anammoxosome, the dedicated intracytoplasmic compartment where anammox catabolism takes place. They give rise to an exceptionally dense membrane, a tight barrier against diffusion. We propose that such a membrane is required to maintain concentration gradients during the exceptionally slow anammox metabolism and to protect the remainder of the cell from the toxic anammox intermediates. Our results further illustrate that microbial membrane lipid structures are far more diverse than previously recognized.