The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase.

Phosphoinositides are spatially restricted membrane signaling molecules. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]--a phosphoinositide that is highly enriched in, and present throughout, the plasma membrane--has been implicated in endocytosis. Trypanosoma brucei has one of the highest known rates of endocytosis, a process it uses to evade the immune system. To determine whether phosphoinositides play a role in endocytosis in this organism, we have identified and characterized one of the enzymes that is responsible for generating PI(4,5)P2. Surprisingly, this phosphoinositide was found to be highly concentrated in the flagellar pocket, the only site of endocytosis and exocytosis in this organism. The enzyme (designated TbPIPKA, annotated as Tb927.10.1620) was present at the neck of the pocket, towards the anterior-end of the parasite. Depletion of TbPIPKA led to depletion of PI(4,5)P2 and enlargement of the pocket, the result of impaired endocytosis. Taken together, these data suggest that TbPIPKA and its product PI(4,5)P2 are important for endocytosis and, consequently, for homeostasis of the flagellar pocket.

The Tetrahymena meiotic chromosome bouquet is organized by centromeres and promotes interhomolog recombination.

In order to form crossovers and to undergo reductional segregation during meiosis, homologous chromosomes must pair. In Tetrahymena, meiotic prophase nuclei elongate immensely, and, within the elongated nucleus, chromosomes are arranged with telomeres assembled at one pole and centromeres at the opposite pole. This organisation is an exaggerated form of the bouquet, a meiotic chromosome arrangement that is widely conserved among eukaryotes. We show that centromere function is crucial for the formation of Tetrahymena's stretched bouquet and, thereby, for homologue pairing. This finding adds to previous reports of the importance of centromeres in chromosome pairing in budding yeast and in Drosophila. Tetrahymena's bouquet is an ataxia telangiectasia- and RAD3-related (ATR)-dependent meiotic DNA damage response that is triggered by meiotic DNA double-strand breaks (DSBs), suggesting that the bouquet is needed for DSB repair. However, in the present study we show that although homologous pairing is impeded in the absence of the bouquet, DSB repair takes place nevertheless. Moreover, recombinational DSB repair, as monitored by bromodeoxyuridine incorporation, takes place only after exit from the bouquet stage. Therefore, we conclude that the bouquet is not required for DSB repair per se, but may be necessary for the alignment of homologous loci in order to promote homologous crossovers over alternative repair pathways.

REvolver: modeling sequence evolution under domain constraints.

Simulating the change of protein sequences over time in a biologically realistic way is fundamental for a broad range of studies with a focus on evolution. It is, thus, problematic that typically simulators evolve individual sites of a sequence identically and independently. More realistic simulations are possible; however, they are often prohibited by limited knowledge concerning site-specific evolutionary constraints or functional dependencies between amino acids. As a consequence, a protein's functional and structural characteristics are rapidly lost in the course of simulated evolution. Here, we present REvolver (www.cibiv.at/software/revolver), a program that simulates protein sequence alteration such that evolutionarily stable sequence characteristics, like functional domains, are maintained. For this purpose, REvolver recruits profile hidden Markov models (pHMMs) for parameterizing site-specific models of sequence evolution in an automated fashion. pHMMs derived from alignments of homologous proteins or protein domains capture information regarding which sequence sites remained conserved over time and where in a sequence insertions or deletions are more likely to occur. Thus, they describe constraints on the evolutionary process acting on these sequences. To demonstrate the performance of REvolver as well as its applicability in large-scale simulation studies, we evolved the entire human proteome up to 1.5 expected substitutions per site. Simultaneously, we analyzed the preservation of Pfam and SMART domains in the simulated sequences over time. REvolver preserved 92% of the Pfam domains originally present in the human sequences. This value drops to 15% when traditional models of amino acid sequence evolution are used. Thus, REvolver represents a significant advance toward a realistic simulation of protein sequence evolution on a proteome-wide scale. Further, REvolver facilitates the simulation of a protein family with a user-defined domain architecture at the root.

FACT: functional annotation transfer between proteins with similar feature architectures.

BACKGROUND: The increasing number of sequenced genomes provides the basis for exploring the genetic and functional diversity within the tree of life. Only a tiny fraction of the encoded proteins undergoes a thorough experimental characterization. For the remainder, bioinformatics annotation tools are the only means to infer their function. Exploiting significant sequence similarities to already characterized proteins, commonly taken as evidence for homology, is the prevalent method to deduce functional equivalence. Such methods fail when homologs are too diverged, or when they have assumed a different function. Finally, due to convergent evolution, functional equivalence is not necessarily linked to common ancestry. Therefore complementary approaches are required to identify functional equivalents. RESULTS: We present the Feature Architecture Comparison Tool http://www.cibiv.at/FACT to search for functionally equivalent proteins. FACT uses the similarity between feature architectures of two proteins, i.e., the arrangements of functional domains, secondary structure elements and compositional properties, as a proxy for their functional equivalence. A scoring function measures feature architecture similarities, which enables searching for functional equivalents in entire proteomes. Our evaluation of 9,570 EC classified enzymes revealed that FACT, using the full feature, set outperformed the existing architecture-based approaches by identifying significantly more functional equivalents as highest scoring proteins. We show that FACT can identify functional equivalents that share no significant sequence similarity. However, when the highest scoring protein of FACT is also the protein with the highest local sequence similarity, it is in 99% of the cases functionally equivalent to the query. We demonstrate the versatility of FACT by identifying a missing link in the yeast glutathione metabolism and also by searching for the human GolgA5 equivalent in Trypanosoma brucei. CONCLUSIONS: FACT facilitates a quick and sensitive search for functionally equivalent proteins in entire proteomes. FACT is complementary to approaches using sequence similarity to identify proteins with the same function. Thus, FACT is particularly useful when functional equivalents need to be identified in evolutionarily distant species, or when functional equivalents are not homologous. The most reliable annotation transfers, however, are achieved when feature architecture similarity and sequence similarity are jointly taken into account.

Tricholoma vaccinum host communication during ectomycorrhiza formation.

The genome sequence of Tricholoma vaccinum was obtained to predict its secretome in order to elucidate communication of T. vaccinum with its host tree spruce (Picea abies) in interkingdom signaling. The most prominent protein domains within the 206 predicted secreted proteins belong to energy and nutrition (52%), cell wall degradation (19%) and mycorrhiza establishment (9%). Additionally, we found small secreted proteins that show typical features of effectors potentially involved in host communication. From the secretome, 22 proteins could be identified, two of which showed higher protein abundances after spruce root exudate exposure, while five were downregulated in this treatment. The changes in T. vaccinum protein excretion with first recognition of the partner were used to identify small secreted proteins with the potential to act as effectors in the mutually beneficial symbiosis. Our observations support the hypothesis of a complex communication network including a cocktail of communication molecules induced long before physical contact of the partners.

Innate immune response to Streptococcus pyogenes depends on the combined activation of TLR13 and TLR2.

Innate immune recognition of the major human-specific Gram-positive pathogen Streptococcus pyogenes is not understood. Here we show that mice employ Toll-like receptor (TLR) 2- and TLR13-mediated recognition of S. pyogenes. These TLR pathways are non-redundant in the in vivo context of animal infection, but are largely redundant in vitro, as only inactivation of both of them abolishes inflammatory cytokine production by macrophages and dendritic cells infected with S. pyogenes. Mechanistically, S. pyogenes is initially recognized in a phagocytosis-independent manner by TLR2 and subsequently by TLR13 upon internalization. We show that the TLR13 response is specifically triggered by S. pyogenes rRNA and that Tlr13-/- cells respond to S. pyogenes infection solely by engagement of TLR2. TLR13 is absent from humans and, remarkably, we find no equivalent route for S. pyogenes RNA recognition in human macrophages. Phylogenetic analysis reveals that TLR13 occurs in all kingdoms but only in few mammals, including mice and rats, which are naturally resistant against S. pyogenes. Our study establishes that the dissimilar expression of TLR13 in mice and humans has functional consequences for recognition of S. pyogenes in these organisms.

In Vivo Effects of Mesenchymal Stromal Cells in Two Patients With Severe Acute Respiratory Distress Syndrome.

UNLABELLED: Mesenchymal stromal cells (MSCs) have been investigated as a treatment for various inflammatory diseases because of their immunomodulatory and reparative properties. However, many basic questions concerning their mechanisms of action after systemic infusion remain unanswered. We performed a detailed analysis of the immunomodulatory properties and proteomic profile of MSCs systemically administered to two patients with severe refractory acute respiratory distress syndrome (ARDS) on a compassionate use basis and attempted to correlate these with in vivo anti-inflammatory actions. Both patients received 2×10(6) cells per kilogram, and each subsequently improved with resolution of respiratory, hemodynamic, and multiorgan failure. In parallel, a decrease was seen in multiple pulmonary and systemic markers of inflammation, including epithelial apoptosis, alveolar-capillary fluid leakage, and proinflammatory cytokines, microRNAs, and chemokines. In vitro studies of the MSCs demonstrated a broad anti-inflammatory capacity, including suppression of T-cell responses and induction of regulatory phenotypes in T cells, monocytes, and neutrophils. Some of these in vitro potency assessments correlated with, and were relevant to, the observed in vivo actions. These experiences highlight both the mechanistic information that can be gained from clinical experience and the value of correlating in vitro potency assessments with clinical effects. The findings also suggest, but do not prove, a beneficial effect of lung protective strategies using adoptively transferred MSCs in ARDS. Appropriate randomized clinical trials are required to further assess any potential clinical efficacy and investigate the effects on in vivo inflammation. SIGNIFICANCE: This article describes the cases of two patients with severe refractory adult respiratory syndrome (ARDS) who failed to improve after both standard life support measures, including mechanical ventilation, and additional measures, including extracorporeal ventilation (i.e., in a heart-lung machine). Unlike acute forms of ARDS (such in the current NIH-sponsored study of mesenchymal stromal cells in ARDS), recovery does not generally occur in such patients.

Zygomycetes, microsporidia, and the evolutionary ancestry of sex determination.

Zygomycetes and their alleged sister taxon, the microsporidia, exclusively share the presence of a cluster of three genes encoding a sugar transporter, a high mobility group (HMG)-type transcription factor, and an RNA helicase. In zygomycetes, the HMG-type transcription factor acts as the sole sex determinant. This intimately ties the evolutionary history of this gene cluster to the evolution of sex determination. Here, we have unraveled the relationships of the two gene clusters by vicariously analyzing the sugar transporters and the RNA helicases. We show that if the two gene clusters share a common ancestry, it dates back to the early days of eukaryotic evolution. As a consequence, the zygomycete MAT locus would be old enough to represent the archetype of fungal and animal sex determination. However, the evolutionary scenario that has to be invoked is complex. An independent assembly of the two clusters deserves therefore consideration. In either case, shared ancestry or convergent evolution, the presence of the gene cluster in microsporidia and in zygomycetes represents at best a plesiomorphy. Hence, it is not phylogenetically informative. A further genome-wide reanalysis of gene order conservation reveals that gene order is not significantly more similar between microsporidia and zygomycetes than between microsporidia and any other fungal taxon or even humans. Consequently, the phylogenetic placement of microsporidia as sister to the zygomycetes needs to be reconsidered.