The <i>N</i>-myristoylome of <i>Trypanosoma cruzi</i>

Protein N-myristoylation is catalysed by N-myristoyltransferase (NMT), an essential and druggable target in Trypanosoma cruzi, the causative agent of Chagas’ disease. Here we have employed whole cell labelling with azidomyristic acid and click chemistry to identify N-myristoylated proteins in different life cycle stages of the parasite. Only minor differences in fluorescent-labelling were observed between the dividing forms (the insect epimastigote and mammalian amastigote stages) and the non-dividing trypomastigote stage. Using a combination of label-free and stable isotope labelling of cells in culture (SILAC) based proteomic strategies in the presence and absence of the NMT inhibitor DDD85646, we identified 56 proteins enriched in at least two out of the three experimental approaches. Of these, 6 were likely to be false positives, with the remaining 50 commencing with amino acids MG at the N-terminus in one or more of the T. cruzi genomes. Most of these are proteins of unknown function (32), with the remainder (18) implicated in a diverse range of critical cellular and metabolic functions such as intracellular transport, cell signalling and protein turnover. In summary, we have established that 0.43–0.46% of the proteome is N-myristoylated in T. cruzi approaching that of other eukaryotic organisms (0.5–1.7%)

Operation and performance of the ATLAS Tile Calorimeter in Run 1

The Tile Calorimeter is the hadron calorimeter covering the central region of the ATLAS experiment at the Large Hadron Collider. Approximately 10,000 photomultipliers collect light from scintillating tiles acting as the active material sandwiched between slabs of steel absorber. This paper gives an overview of the calorimeter’s performance during the years 2008–2012 using cosmic-ray muon events and proton–proton collision data at centre-of-mass energies of 7 and 8TeV with a total integrated luminosity of nearly 30 fb−1. The signal reconstruction methods, calibration systems as well as the detector operation status are presented. The energy and time calibration methods performed excellently, resulting in good stability of the calorimeter response under varying conditions during the LHC Run 1. Finally, the Tile Calorimeter response to isolated muons and hadrons as well as to jets from proton–proton collisions is presented. The results demonstrate excellent performance in accord with specifications mentioned in the Technical Design Report

A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes

Sex, Subdivision, and Domestic Dispersal of Trypanosoma cruzi Lineage I in Southern Ecuador

Trypanosoma cruzi is transmitted by blood sucking insects known as triatomines. This protozoan parasite commonly infects wild and domestic mammals in South and Central America. However, triatomines also transmit the parasite to people, and human infection with T. cruzi is known as Chagas disease, a major public health concern in Latin America. Understanding the complex dynamics of parasite spread between wild and domestic environments is essential to design effective control measures to prevent the spread of Chagas disease. Here we describe T. cruzi genetic diversity and population dynamics in southern Ecuador. Our findings indicate that the parasite circulates in two largely independent cycles: one corresponding to the sylvatic environment and one related to the domestic/peridomestic environment. Furthermore, our data indicate that human activity might promote parasite dispersal among communties. This information is the key for the design of control programmes in Southern Ecuador. Finally, we have encountered evidence of a sexual reproductive mode in the domestic T. cruzi population, which constitutes a new and intriguing finding with regards to the biology of this parasite

Conserved BK Channel-Protein Interactions Reveal Signals Relevant to Cell Death and Survival

The large-conductance Ca2+-activated K+ (BK) channel and its β-subunit underlie tuning in non-mammalian sensory or hair cells, whereas in mammals its function is less clear. To gain insights into species differences and to reveal putative BK functions, we undertook a systems analysis of BK and BK-Associated Proteins (BKAPS) in the chicken cochlea and compared these results to other species. We identified 110 putative partners from cytoplasmic and membrane/cytoskeletal fractions, using a combination of coimmunoprecipitation, 2-D gel, and LC-MS/MS. Partners included 14-3-3γ, valosin-containing protein (VCP), stathmin (STMN), cortactin (CTTN), and prohibitin (PHB), of which 16 partners were verified by reciprocal coimmunoprecipitation. Bioinformatics revealed binary partners, the resultant interactome, subcellular localization, and cellular processes. The interactome contained 193 proteins involved in 190 binary interactions in subcellular compartments such as the ER, mitochondria, and nucleus. Comparisons with mice showed shared hub proteins that included N-methyl-D-aspartate receptor (NMDAR) and ATP-synthase. Ortholog analyses across six species revealed conserved interactions involving apoptosis, Ca2+ binding, and trafficking, in chicks, mice, and humans. Functional studies using recombinant BK and RNAi in a heterologous expression system revealed that proteins important to cell death/survival, such as annexinA5, γ-actin, lamin, superoxide dismutase, and VCP, caused a decrease in BK expression. This revelation led to an examination of specific kinases and their effectors relevant to cell viability. Sequence analyses of the BK C-terminus across 10 species showed putative binding sites for 14-3-3, RAC-α serine/threonine-protein kinase 1 (Akt), glycogen synthase kinase-3β (GSK3β) and phosphoinositide-dependent kinase-1 (PDK1). Knockdown of 14-3-3 and Akt caused an increase in BK expression, whereas silencing of GSK3β and PDK1 had the opposite effect. This comparative systems approach suggests conservation in BK function across different species in addition to novel functions that may include the initiation of signals relevant to cell death/survival

Shotgun Sequencing Analysis of Trypanosoma cruzi I Sylvio X10/1 and Comparison with T. cruzi VI CL Brener

Trypanosoma cruzi is the causative agent of Chagas disease, which affects more than 9 million people in Latin America. We have generated a draft genome sequence of the TcI strain Sylvio X10/1 and compared it to the TcVI reference strain CL Brener to identify lineage-specific features. We found virtually no differences in the core gene content of CL Brener and Sylvio X10/1 by presence/absence analysis, but 6 open reading frames from CL Brener were missing in Sylvio X10/1. Several multicopy gene families, including DGF, mucin, MASP and GP63 were found to contain substantially fewer genes in Sylvio X10/1, based on sequence read estimations. 1,861 small insertion-deletion events and 77,349 nucleotide differences, 23% of which were non-synonymous and associated with radical amino acid changes, further distinguish these two genomes. There were 336 genes indicated as under positive selection, 145 unique to T. cruzi in comparison to T. brucei and Leishmania. This study provides a framework for further comparative analyses of two major T. cruzi lineages and also highlights the need for sequencing more strains to understand fully the genomic composition of this parasite

Analyses of 32 Loci Clarify Phylogenetic Relationships among Trypanosoma cruzi Lineages and Support a Single Hybridization prior to Human Contact

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease, a major health problem in Latin America. The genetic diversity of this parasite has been traditionally divided in two major groups: T. cruzi I and II, which can be further divided in six major genetic subdivisions (subgroups TcI-TcVI). T. cruzi I and II seem to differ in important biological characteristics, and are thought to represent a natural division relevant for epidemiological studies and development of prophylaxis. Having a correct reconstruction of the evolutionary history of T. cruzi is essential for understanding the potential connection between the genetic and phenotypic variability of T. cruzi with the different manifestations of Chagas disease. Here we present results from a comprehensive phylogenetic analysis of T. cruzi using more than 26 Kb of aligned sequence data. We show strong evidence that T. cruzi II (TcII-VI) is not a natural evolutionary group but a paraphyletic lineage and that all major lineages of T. cruzi evolved recently (<3 million years ago [mya]). Furthermore, the sequence data is consistent with one major hybridization event having occurred in this species recently (< 1 mya) but well before T. cruzi entered in contact with humans in South America

The Short Non-Coding Transcriptome of the Protozoan Parasite Trypanosoma cruzi

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16–61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95–98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3′ end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes

Computational Methods for Protein Identification from Mass Spectrometry Data

Protein identification using mass spectrometry is an indispensable computational tool in the life sciences. A dramatic increase in the use of proteomic strategies to understand the biology of living systems generates an ongoing need for more effective, efficient, and accurate computational methods for protein identification. A wide range of computational methods, each with various implementations, are available to complement different proteomic approaches. A solid knowledge of the range of algorithms available and, more critically, the accuracy and effectiveness of these techniques is essential to ensure as many of the proteins as possible, within any particular experiment, are correctly identified. Here, we undertake a systematic review of the currently available methods and algorithms for interpreting, managing, and analyzing biological data associated with protein identification. We summarize the advances in computational solutions as they have responded to corresponding advances in mass spectrometry hardware. The evolution of scoring algorithms and metrics for automated protein identification are also discussed with a focus on the relative performance of different techniques. We also consider the relative advantages and limitations of different techniques in particular biological contexts. Finally, we present our perspective on future developments in the area of computational protein identification by considering the most recent literature on new and promising approaches to the problem as well as identifying areas yet to be explored and the potential application of methods from other areas of computational biology