A comprehensive assessment of N-terminal signal peptides prediction methods

Background: Amino-terminal signal peptides (SPs) are short regions that guide the targeting of secretory proteins to the correct subcellular compartments in the cell. They are cleaved off upon the passenger protein reaching its destination. The explosive growth in sequencing technologies has led to the deposition of vast numbers of protein sequences necessitating rapid functional annotation techniques, with subcellular localization being a key feature. Of the myriad software prediction tools developed to automate the task of assigning the SP cleavage site of these new sequences, we review here, the performance and reliability of commonly used SP prediction tools. Results: The available signal peptide data has been manually curated and organized into three datasets representing eukaryotes, Gram-positive and Gram-negative bacteria. These datasets are used to evaluate thirteen prediction tools that are publicly available. SignalP (both the HMM and ANN versions) maintains consistency and achieves the best overall accuracy in all three benchmarking experiments, ranging from 0.872 to 0.914 although other prediction tools are narrowing the performance gap. Conclusion: The majority of the tools evaluated in this study encounter no difficulty in discriminating between secretory and non-secretory proteins. The challenge clearly remains with pinpointing the correct SP cleavage site. The composite scoring schemes employed by SignalP may help to explain its accuracy. Prediction task is divided into a number of separate steps, thus allowing each score to tackle a particular aspect of the prediction.12 page(s

The Proteomics of N-terminal Methionine Cleavage

Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1′), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (\u3e120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1′ were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues at P2′, P3′, and P4′ strongly slowed the reaction, and some proteins with Val and Thr at P1′ could not undergo Met cleavage. These in vitro data were fully consistent with data for 862 E. coli proteins with known N-terminal sequences in vivo. The specificity sites were found to be identical to those for the other type of MAPs, MAP2s, and a dedicated prediction tool for Met cleavage is now available. Taking into account the rules of MAP cleavage and leader peptide removal, the N termini of all proteins were predicted from the annotated genome and compared with data obtained in vivo. This analysis showed that proteins displaying N-Met cleavage are overrepresented in vivo. We conclude that protein secretion involving leader peptide cleavage is more frequent than generally thought

Urine peptidomic biomarkers for diagnosis of patients with systematic lupus erythematosus

Background: Systematic lupus erythematosus (SLE) is characterized with various complications which can cause serious organ damage in the human body. Despite the significant improvements in disease management of SLE patients, the non-invasive diagnosis is entirely missing. In this study, we used urinary peptidomic biomarkers for early diagnosis of disease onset to improve patient risk stratification, vital for effective drug treatment. Methods: Urine samples from patients with SLE, lupus nephritis (LN) and healthy controls (HCs) were analyzed using capillary electrophoresis coupled to mass spectrometry (CE-MS) for state-of-the-art biomarker discovery. Results: A biomarker panel made up of 65 urinary peptides was developed that accurately discriminated SLE without renal involvement from HC patients. The performance of the SLE-specific panel was validated in a multicentric independent cohort consisting of patients without SLE but with different renal disease and LN. This resulted in an area under the receiver operating characteristic (ROC) curve (AUC) of 0.80 (p < 0.0001, 95% confidence interval (CI) 0.65–0.90) corresponding to a sensitivity and a specificity of 83% and 73%, respectively. Based on the end terminal amino acid sequences of the biomarker peptides, an in silico methodology was used to identify the proteases that were up or down-regulated. This identified matrix metalloproteinases (MMPs) as being mainly responsible for the peptides fragmentation. Conclusions: A laboratory-based urine test was successfully established for early diagnosis of SLE patients. Our approach determined the activity of several proteases and provided novel molecular information that could potentially influence treatment efficacy

Nezara viridula (Hemiptera: Pentatomidae) transcriptomic analysis and neuropeptidomics

Stinkbugs (Hemiptera: Pentatomidae) are of major economic importance as pest of crops. Among the species composing the stinkbug complex, Nezara viridula is one of the most abundant in Brazil, Argentina and the Southern USA. However, this species has been poorly characterized at the genetic and physiological level. Here we sequenced and analyzed the complete transcriptome of N. viridula male and female adults. We identified neuropeptide precursor genes and G-protein coupled receptors for neuropeptides in this transcriptome. Mature neuropeptides were identified in N. viridula brain extracts by liquid chromatography-tandem mass spectrometry. We also analyzed the neuropeptide precursor complement in the genome sequence of Halyomorpha halys, another pentatomid of economic relevance. We compared the results in both pentatomids with the well-characterized neuropeptide repertoire from the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae). We identified both group-specific features (which could be related to the different feeding habits) and similarities that could be characteristic of Heteroptera. This work contributes to a deeper knowledge of the genetic information of these pests, with a focus on neuroendocrine system characterization.Fil: Lavore, Andres Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Pérez Gianmarco, Lucila Maité. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Esponda Behrens, Natalia Irene. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Palacio, Victorio Gabriel. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Catalano, María Inés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); ArgentinaFil: Rivera Pomar, Rolando. Universidad Nacional del Noroeste de la Provincia de Buenos Aires. Centro de Bioinvestigaciones (Sede Pergamino); Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; ArgentinaFil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Ribosome signatures aid bacterial translation initiation site identification

Background: While methods for annotation of genes are increasingly reliable, the exact identification of translation initiation sites remains a challenging problem. Since the N-termini of proteins often contain regulatory and targeting information, developing a robust method for start site identification is crucial. Ribosome profiling reads show distinct patterns of read length distributions around translation initiation sites. These patterns are typically lost in standard ribosome profiling analysis pipelines, when reads from footprints are adjusted to determine the specific codon being translated. Results: Utilising these signatures in combination with nucleotide sequence information, we build a model capable of predicting translation initiation sites and demonstrate its high accuracy using N-terminal proteomics. Applying this to prokaryotic translatomes, we re-annotate translation initiation sites and provide evidence of N-terminal truncations and extensions of previously annotated coding sequences. These re-annotations are supported by the presence of structural and sequence-based features next to N-terminal peptide evidence. Finally, our model identifies 61 novel genes previously undiscovered in the Salmonella enterica genome. Conclusions: Signatures within ribosome profiling read length distributions can be used in combination with nucleotide sequence information to provide accurate genome-wide identification of translation initiation sites

An extra dimension in protein tagging by quantifying universal proteotypic peptides using targeted proteomics

The use of protein tagging to facilitate detailed characterization of target proteins has not only revolutionized cell biology, but also enabled biochemical analysis through efficient recovery of the protein complexes wherein the tagged proteins reside. The endogenous use of these tags for detailed protein characterization is widespread in lower organisms that allow for efficient homologous recombination. With the recent advances in genome engineering, tagging of endogenous proteins is now within reach for most experimental systems, including mammalian cell lines cultures. In this work, we describe the selection of peptides with ideal mass spectrometry characteristics for use in quantification of tagged proteins using targeted proteomics. We mined the proteome of the hyperthermophile Pyrococcus furiosus to obtain two peptides that are unique in the proteomes of all known model organisms (proteotypic) and allow sensitive quantification of target proteins in a complex background. By combining these 'Proteotypic peptides for Quantification by SRM' (PQS peptides) with epitope tags, we demonstrate their use in co-immunoprecipitation experiments upon transfection of protein pairs, or after introduction of these tags in the endogenous proteins through genome engineering. Endogenous protein tagging for absolute quantification provides a powerful extra dimension to protein analysis, allowing the detailed characterization of endogenous proteins

FFAS server: novel features and applications.

The Fold and Function Assignment System (FFAS) server [Jaroszewski et al. (2005) FFAS03: a server for profile-profile sequence alignments. Nucleic Acids Research, 33, W284-W288] implements the algorithm for protein profile-profile alignment introduced originally in [Rychlewski et al. (2000) Comparison of sequence profiles. Strategies for structural predictions using sequence information. Protein Science: a Publication of the Protein Society, 9, 232-241]. Here, we present updates, changes and novel functionality added to the server since 2005 and discuss its new applications. The sequence database used to calculate sequence profiles was enriched by adding sets of publicly available metagenomic sequences. The profile of a user's protein can now be compared with ∼20 additional profile databases, including several complete proteomes, human proteins involved in genetic diseases and a database of microbial virulence factors. A newly developed interface uses a system of tabs, allowing the user to navigate multiple results pages, and also includes novel functionality, such as a dotplot graph viewer, modeling tools, an improved 3D alignment viewer and links to the database of structural similarities. The FFAS server was also optimized for speed: running times were reduced by an order of magnitude. The FFAS server, http://ffas.godziklab.org, has no log-in requirement, albeit there is an option to register and store results in individual, password-protected directories. Source code and Linux executables for the FFAS program are available for download from the FFAS server

The Roles of Gene Duplication, Gene Conversion and Positive Selection in Rodent \u3ci\u3eEsp\u3c/i\u3e and \u3ci\u3eMup\u3c/i\u3e Pheromone Gene Families with Comparison to the \u3ci\u3eAbp\u3c/i\u3e Family

Three proteinaceous pheromone families, the androgen-binding proteins (ABPs), the exocrine-gland secreting peptides (ESPs) and the major urinary proteins (MUPs) are encoded by large gene families in the genomes of Mus musculus and Rattus norvegicus. We studied the evolutionary histories of the Mup and Esp genes and compared them with what is known about the Abp genes. Apparently gene conversion has played little if any role in the expansion of the mouse Class A and Class B Mup genes and pseudogenes, and the rat Mups. By contrast, we found evidence of extensive gene conversion in many Esp genes although not in all of them. Our studies of selection identified at least two amino acid sites in β-sheets as having evolved under positive selection in the mouse Class A and Class B MUPs and in rat MUPs. We show that selection may have acted on the ESPs by determining Ka/Ks for Exon 3 sequences with and without the converted sequence segment. While it appears that purifying selection acted on the ESP signal peptides, the secreted portions of the ESPs probably have undergone much more rapid evolution. When the inner gene converted fragment sequences were removed, eleven Esp paralogs were present in two or more pairs with Ka/Ks \u3e1.0 and thus we propose that positive selection is detectable by this means in at least some mouse Esp paralogs. We compare and contrast the evolutionary histories of all three mouse pheromone gene families in light of their proposed functions in mouse communication