Mitochondrial damage by α-synuclein causes cell death in human dopaminergic neurons.

Evolving concepts on Parkinson's disease (PD) pathology suggest that α-synuclein (aSYN) promote dopaminergic neuron dysfunction and death through accumulating in the mitochondria. However, the consequence of mitochondrial aSYN localisation on mitochondrial structure and bioenergetic functions in neuronal cells are poorly understood. Therefore, we investigated deleterious effects of mitochondria-targeted aSYN in differentiated human dopaminergic neurons in comparison with wild-type (WT) aSYN overexpression and corresponding EGFP (enhanced green fluorescent protein)-expressing controls. Mitochondria-targeted aSYN enhanced mitochondrial reactive oxygen species (ROS) formation, reduced ATP levels and showed severely disrupted structure and function of the dendritic neural network, preceding neuronal death. Transmission electron microscopy illustrated distorted cristae and many fragmented mitochondria in response to WT-aSYN overexpression, and a complete loss of cristae structure and massively swollen mitochondria in neurons expressing mitochondria-targeted aSYN. Further, the analysis of mitochondrial bioenergetics in differentiated dopaminergic neurons, expressing WT or mitochondria-targeted aSYN, elicited a pronounced impairment of mitochondrial respiration. In a pharmacological compound screening, we found that the pan-caspase inhibitors QVD and zVAD-FMK, and a specific caspase-1 inhibitor significantly prevented aSYN-induced cell death. In addition, the caspase inhibitor QVD preserved mitochondrial function and neuronal network activity in the human dopaminergic neurons overexpressing aSYN. Overall, our findings indicated therapeutic effects by caspase-1 inhibition despite aSYN-mediated alterations in mitochondrial morphology and function.

Legionella pneumophila infection activates bystander cells differentially by bacterial and host cell vesicles.

Extracellular vesicles from eukaryotic cells and outer membrane vesicles (OMVs) released from gram-negative bacteria have been described as mediators of pathogen-host interaction and intercellular communication. Legionella pneumophila (L. pneumophila) is a causative agent of severe pneumonia. The differential effect of bacterial and host cell vesicles in L. pneumophila infection is unknown so far. We infected THP-1-derived or primary human macrophages with L. pneumophila and isolated supernatant vesicles by differential centrifugation. We observed an increase of exosomes in the 100 k pellet by nanoparticle tracking analysis, electron microscopy, and protein markers. This fraction additionally contained Legionella LPS, indicating also the presence of OMVs. In contrast, vesicles in the 16 k pellet, representing microparticles, decreased during infection. The 100 k vesicle fraction activated uninfected primary human alveolar epithelial cells, A549 cells, and THP-1 cells. Epithelial cell activation was reduced by exosome depletion (anti-CD63, or GW4869), or blocking of IL-1ß in the supernatant. In contrast, the response of THP-1 cells to vesicles was reduced by a TLR2-neutralizing antibody, UV-inactivation of bacteria, or - partially - RNase-treatment of vesicles. Taken together, we found that during L. pneumophila infection, neighbouring epithelial cells were predominantly activated by exosomes and cytokines, whereas myeloid cells were activated by bacterial OMVs.

The Human Antimicrobial Protein Bactericidal/Permeability-Increasing Protein (BPI) Inhibits the Infectivity of Influenza A Virus.

In addition to their well-known antibacterial activity some antimicrobial peptides and proteins (AMPs) display also antiviral effects. A 27 aa peptide from the N-terminal part of human bactericidal/permeability-increasing protein (BPI) previously shown to harbour antibacterial activity inhibits the infectivity of multiple Influenza A virus strains (H1N1, H3N2 and H5N1) the causing agent of the Influenza pneumonia. In contrast, the homologous murine BPI-peptide did not show activity against Influenza A virus. In addition human BPI-peptide inhibits the activation of immune cells mediated by Influenza A virus. By changing the human BPI-peptide to the sequence of the mouse homologous peptide the antiviral activity was completely abolished. Furthermore, the human BPI-peptide also inhibited the pathogenicity of the Vesicular Stomatitis Virus but failed to interfere with HIV and measles virus. Electron microscopy indicate that the human BPI-peptide interferes with the virus envelope and at high concentrations was able to destroy the particles completely.

The evolution of eukaryotic cells from the perspective of peroxisomes: phylogenetic analyses of peroxisomal beta-oxidation enzymes support mitochondria-first models of eukaryotic cell evolution.

Beta-oxidation of fatty acids and detoxification of reactive oxygen species are generally accepted as being fundamental functions of peroxisomes. Additionally, these pathways might have been the driving force favoring the selection of this compartment during eukaryotic evolution. Here we performed phylogenetic analyses of enzymes involved in beta-oxidation of fatty acids in Bacteria, Eukaryota, and Archaea. These imply an alpha-proteobacterial origin for three out of four enzymes. By integrating the enzymes' history into the contrasting models on the origin of eukaryotic cells, we conclude that peroxisomes most likely evolved non-symbiotically and subsequent to the acquisition of mitochondria in an archaeal host cell.

The periplastidal compartment: a naturally minimized eukaryotic cytoplasm.

Many important algae groups like diatoms, dinoflagellates and 'kelp' but also apicomplexan parasites evolved in secondary endosymbiosis. Here, a eukaryote-eukaryote endosymbiosis created chimeric cells, in which a eukaryotic symbiont was reduced to a complex plastid. Although having lost nearly all of the eukaryotic compartments of the symbiont, a tiny lumen representing the remnant of the cytoplasm of the symbiont is still present in most of these organisms. This compartment, the periplastidal compartment, shows different degrees of reductions as in two algal groups the former nucleus is still present in a minimized form, called nucleomorph, whereas most others have lost the genetic system completely. Thus, the natural reduction of eukaryotic cytoplasms can be studied in terms of evolution and functionality, giving additionally advices for the design of synthetic minimized compartments.

Massively convergent evolution for ribosomal protein gene content in plastid and mitochondrial genomes.

Plastid and mitochondrial genomes have undergone parallel evolution to encode the same functional set of genes. These encode conserved protein components of the electron transport chain in their respective bioenergetic membranes and genes for the ribosomes that express them. This highly convergent aspect of organelle genome evolution is partly explained by the redox regulation hypothesis, which predicts a separate plastid or mitochondrial location for genes encoding bioenergetic membrane proteins of either photosynthesis or respiration. Here we show that convergence in organelle genome evolution is far stronger than previously recognized, because the same set of genes for ribosomal proteins is independently retained by both plastid and mitochondrial genomes. A hitherto unrecognized selective pressure retains genes for the same ribosomal proteins in both organelles. On the Escherichia coli ribosome assembly map, the retained proteins are implicated in 30S and 50S ribosomal subunit assembly and initial rRNA binding. We suggest that ribosomal assembly imposes functional constraints that govern the retention of ribosomal protein coding genes in organelles. These constraints are subordinate to redox regulation for electron transport chain components, which anchor the ribosome to the organelle genome in the first place. As organelle genomes undergo reduction, the rRNAs also become smaller. Below size thresholds of approximately 1,300 nucleotides (16S rRNA) and 2,100 nucleotides (26S rRNA), all ribosomal protein coding genes are lost from organelles, while electron transport chain components remain organelle encoded as long as the organelles use redox chemistry to generate a proton motive force.

Two small GTPases act in concert with the bactofilin cytoskeleton to regulate dynamic bacterial cell polarity.

Cell polarity is essential for many bacterial activities, but the mechanisms responsible for its establishment are poorly understood. In Myxococcus xanthus, the type IV pili (T4P) motor ATPases PilB and PilT localize to opposite cell poles and switch poles during cellular reversals. We demonstrate that polar localization of PilB and PilT depends on the small GTPase SofG and BacP, a bactofilin cytoskeletal protein. Polymeric BacP localizes in both subpolar regions. SofG interacts directly with polymeric BacP and associates with one of these patches, forming a cluster that shuttles to the pole to establish localization of PilB and PilT at the same pole. Next, the small GTPase MglA sorts PilB and PilT to opposite poles to establish their correct polarity. During reversals, the Frz chemosensory system induces the inversion of PilB and PilT polarity. Thus, three hierarchically organized systems function in a cascade to regulate dynamic bacterial cell polarity.

Knockout of the abundant Trichomonas vaginalis hydrogenosomal membrane protein TvHMP23 increases hydrogenosome size but induces no compensatory up-regulation of paralogous copies.

The Trichomonas vaginalis genome encodes up to 60000 genes, many of which stem from genome duplication events. Paralogous copies thus accompany most T. vaginalis genes, a phenomenon that limits genetic manipulation. We characterized one of the parasite's most abundant hydrogenosomal membrane proteins, TvHMP23, which is phylogenetically distinct from canonical metabolite carriers, and which localizes to the inner hydrogenosomal membrane as shown through sub-organellar fractionation and protease protection assays. Knockout of Tvhmp23 through insertion of the selectable neomycin marker led to a size increase of hydrogenosomes, the first knockout-induced phenotypes reported for Trichomonas, but no growth impairment. The transcriptional response of its four paralogous copies then analyzed revealed that they are not up-regulated, and hence do not compensate for the Tvhmp23 knockout.

Novosphingobium fuchskuhlense sp. nov., isolated from the north-east basin of Lake Grosse Fuchskuhle.

A yellow pigmented, gram-negative, rod-shaped bacterium designated FNE08-7(T) was isolated from subsurface water of the north-east basin of the bog lake Grosse Fuchskuhle (Brandenburg, Germany). A first analysis of the nearly full-length 16S rRNA gene sequence analysis including environmental 16S rRNA gene sequences derived from freshwater ecosystems showed that strain FNE08-7(T) is the first cultured representative, to our knowledge, of the freshwater tribe Novo-A2. Further analysis indicates highest 16S rRNA gene sequence similarities to the type strains of Novosphingobium stygium (98.0 %) and Novosphingobium taihuense (97.4 %) and between 94.0 % and 96.9 % sequence similarity to other members of the genus Novosphingobium. Reconstruction of phylogenetic trees showed that strain FNE08-7(T) formed a distinct cluster with the type strains of N. stygium and N. taihuense supported by high bootstrap values. DNA-DNA hybridization of strain FNE08-7(T) with N. stygium SMCC B0712(T) and N. taihuense DSM 17507(T) revealed low similarity values of 18.4 % (reciprocal: 11.4 %) and 23.1 % (reciprocal: 54.2 %), respectively. The predominant fatty acid of the isolate is C(18 : 1)ω7c (56.4 %) and two characteristic 2-hydroxy fatty acids, C(14 : 0) 2-OH (16.5 %) and C(15 : 0) 2-OH (3.3 %) occur. Ubiquinone Q-10 is the major respiratory quinone. The predominant polar lipids are phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, sphingoglycolipid, phosphatidylcholine and minor amounts of diphosphatidylglycerol. Spermidine is the predominant polyamine. Characterization by genotypic, chemotaxonomic and phenotypic analysis indicate that strain FNE08-7(T) represents a novel species of the genus Novosphingobium within the Alphaproteobacteria. Therefore, we propose the species Novosphingobium fuchskuhlense sp. nov., with FNE08-7(T) ( = DSM 25065(T) = CCM 7978(T) = CCUG 61508(T)) as the type strain.

In vivo function of Tic22, a protein import component of the intermembrane space of chloroplasts.

Preprotein import into chloroplasts depends on macromolecular machineries in the outer and inner chloroplast envelope membrane (TOC and TIC). It was suggested that both machineries are interconnected by components of the intermembrane space (IMS). That is, amongst others, Tic22, of which two closely related isoforms exist in Arabidopsis thaliana, namely atTic22-III and atTic22-IV. We investigated the function of Tic22 in vivo by analyzing T-DNA insertion lines of the corresponding genes. While the T-DNA insertion in the individual genes caused only slight defects, a double mutant of both isoforms showed retarded growth, a pale phenotype under high-light conditions, a reduced import rate, and a reduction in the photosynthetic performance of the plants. The latter is supported by changes in the metabolite content of mutant plants when compared to wild-type. Thus, our results support the notion that Tic22 is directly involved in chloroplast preprotein import and might point to a particular importance of Tic22 in chloroplast biogenesis at times of high import rates.

Novosphingobium aquaticum sp. nov., isolated from the humic-matter-rich bog lake Grosse Fuchskuhle.

A yellow-pigmented, Gram-negative rod, designated FNE08-86(T), was isolated from subsurface water of the humic-matter-rich and almost-neutral north-east basin of the experimentally divided bog lake Grosse Fuchskuhle (Brandenburg, Germany). Analysis of the nearly full-length 16S rRNA gene sequence showed the highest 16S rRNA gene sequence similarity with Novosphingobium rosa IAM 14222(T) (96.3 %). Sequence similarities with all other members of the genus Novosphingobium species were <96 %, but phylogenetic tree construction clearly showed the placement of strain FNE08-86(T) within the genus Novosphingobium. The predominant fatty acids were C18 : 1ω7c and C16 : 0, and only a single 2-hydroxy fatty acid, C14 : 0 2-OH, was detected. The polar lipid profile revealed phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine as major compounds, with smaller amounts of sphingoglycolipid, phosphatidylmonomethylethanolamine, diphosphatidylglycerol and several unidentified lipids. In the quinone system ubiquinone Q-10 was predominant and in the polyamine pattern spermidine was predominant. Characterization by genotypic, chemotaxonomic and phenotypic analysis indicated that strain FNE08-86(T) represents a novel species of the genus Novosphingobium, for which we propose the name Novosphingobium aquaticum sp. nov. (type strain FNE08-86(T) = DSM 25088(T) = CCM 7983(T)).

General protein diffusion barriers create compartments within bacterial cells.

In eukaryotes, the differentiation of cellular extensions such as cilia or neuronal axons depends on the partitioning of proteins to distinct plasma membrane domains by specialized diffusion barriers. However, examples of this compartmentalization strategy are still missing for prokaryotes, although complex cellular architectures are also widespread among this group of organisms. This study reveals the existence of a protein-mediated membrane diffusion barrier in the stalked bacterium Caulobacter crescentus. We show that the Caulobacter cell envelope is compartmentalized by macromolecular complexes that prevent the exchange of both membrane and soluble proteins between the polar stalk extension and the cell body. The barrier structures span the cross-sectional area of the stalk and comprise at least four proteins that assemble in a cell-cycle-dependent manner. Their presence is critical for cellular fitness because they minimize the effective cell volume, allowing faster adaptation to environmental changes that require de novo synthesis of envelope proteins.

A single peroxisomal targeting signal mediates matrix protein import in diatoms.

Peroxisomes are single membrane bound compartments. They are thought to be present in almost all eukaryotic cells, although the bulk of our knowledge about peroxisomes has been generated from only a handful of model organisms. Peroxisomal matrix proteins are synthesized cytosolically and posttranslationally imported into the peroxisomal matrix. The import is generally thought to be mediated by two different targeting signals. These are respectively recognized by the two import receptor proteins Pex5 and Pex7, which facilitate transport across the peroxisomal membrane. Here, we show the first in vivo localization studies of peroxisomes in a representative organism of the ecologically relevant group of diatoms using fluorescence and transmission electron microscopy. By expression of various homologous and heterologous fusion proteins we demonstrate that targeting of Phaeodactylum tricornutum peroxisomal matrix proteins is mediated only by PTS1 targeting signals, also for proteins that are in other systems imported via a PTS2 mode of action. Additional in silico analyses suggest this surprising finding may also apply to further diatoms. Our data suggest that loss of the PTS2 peroxisomal import signal is not reserved to Caenorhabditis elegans as a single exception, but has also occurred in evolutionary divergent organisms. Obviously, targeting switching from PTS2 to PTS1 across different major eukaryotic groups might have occurred for different reasons. Thus, our findings question the widespread assumption that import of peroxisomal matrix proteins is generally mediated by two different targeting signals. Our results implicate that there apparently must have been an event causing the loss of one targeting signal even in the group of diatoms. Different possibilities are discussed that indicate multiple reasons for the detected targeting switching from PTS2 to PTS1.

Making new out of old: recycling and modification of an ancient protein translocation system during eukaryotic evolution. Mechanistic comparison and phylogenetic analysis of ERAD, SELMA and the peroxisomal importomer.

At first glance the three eukaryotic protein translocation machineries--the ER-associated degradation (ERAD) transport apparatus of the endoplasmic reticulum, the peroxisomal importomer and SELMA, the pre-protein translocator of complex plastids--appear quite different. However, mechanistic comparisons and phylogenetic analyses presented here suggest that all three translocation machineries share a common ancestral origin, which highlights the recycling of pre-existing components as an effective evolutionary driving force. Editor's suggested further reading in BioEssays ERAD ubiquitin ligases Abstract.

Identification and characterization of NAGNAG alternative splicing in the moss Physcomitrella patens.

BACKGROUND: Alternative splicing (AS) involving tandem acceptors that are separated by three nucleotides (NAGNAG) is an evolutionarily widespread class of AS, which is well studied in Homo sapiens (human) and Mus musculus (mouse). It has also been shown to be common in the model seed plants Arabidopsis thaliana and Oryza sativa (rice). In one of the first studies involving sequence-based prediction of AS in plants, we performed a genome-wide identification and characterization of NAGNAG AS in the model plant Physcomitrella patens, a moss. RESULTS: Using Sanger data, we found 295 alternatively used NAGNAG acceptors in P. patens. Using 31 features and training and test datasets of constitutive and alternative NAGNAGs, we trained a classifier to predict the splicing outcome at NAGNAG tandem splice sites (alternative splicing, constitutive at the first acceptor, or constitutive at the second acceptor). Our classifier achieved a balanced specificity and sensitivity of >or= 89%. Subsequently, a classifier trained exclusively on data well supported by transcript evidence was used to make genome-wide predictions of NAGNAG splicing outcomes. By generation of more transcript evidence from a next-generation sequencing platform (Roche 454), we found additional evidence for NAGNAG AS, with altogether 664 alternative NAGNAGs being detected in P. patens using all currently available transcript evidence. The 454 data also enabled us to validate the predictions of the classifier, with 64% (80/125) of the well-supported cases of AS being predicted correctly. CONCLUSION: NAGNAG AS is just as common in the moss P. patens as it is in the seed plants A. thaliana and O. sativa (but not conserved on the level of orthologous introns), and can be predicted with high accuracy. The most informative features are the nucleotides in the NAGNAG and in its immediate vicinity, along with the splice sites scores, as found earlier for NAGNAG AS in animals. Our results suggest that the mechanism behind NAGNAG AS in plants is similar to that in animals and is largely dependent on the splice site and its immediate neighborhood.

Protein targeting into secondary plastids.

Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus-encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus-encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre-existing ones to new requirements in secondary plastids.

Complementation of a phycocyanin-bilin lyase from Synechocystis sp. PCC 6803 with a nucleomorph-encoded open reading frame from the cryptophyte Guillardia theta.

BACKGROUND: Cryptophytes are highly compartmentalized organisms, expressing a secondary minimized eukaryotic genome in the nucleomorph and its surrounding remnant cytoplasm, in addition to the cell nucleus, the mitochondrion and the plastid. Because the members of the nucleomorph-encoded proteome may contribute to essential cellular pathways, elucidating nucleomorph-encoded functions is of utmost interest. Unfortunately, cryptophytes are inaccessible for genetic transformations thus far. Therefore the functions of nucleomorph-encoded proteins must be elucidated indirectly by application of methods in genetically accessible organisms. RESULTS: Orf222, one of the uncharacterized nucleomorph-specific open reading frames of the cryptophyte Guillardia theta, shows homology to slr1649 of Synechocystis sp. PCC 6803. Recently a further homolog from Synechococcus sp. PCC 7002 was characterized to encode a phycocyanin-beta155-bilin lyase. Here we show by insertion mutagenesis that the Synechocystis sp. PCC 6803 slr1649-encoded protein also acts as a bilin lyase, and additionally contributes to linker attachment and/or stability of phycobilisomes. Finally, our results indicate that the phycocyanin-beta155-bilin lyase of Synechocystis sp. PCC 6803 can be complemented in vivo by the nucleomorph-encoded open reading frame orf222. CONCLUSION: Our data show that the loss of phycocyanin-lyase function causes pleiotropic effects in Synechocystis sp. PCC 6803 and indicate that after separating from a common ancestor protein, the phycoerythrin lyase from Guillardia theta has retained its capacity to couple a bilin group to other phycobiliproteins. This is a further, unexpected example of the universality of phycobiliprotein lyases.