scholarly journals Evaluation of signal peptide prediction algorithms for identification of mycobacterial signal peptides using sequence data from proteomic methods

Microbiology ◽  
2009 ◽  
Vol 155 (7) ◽  
pp. 2375-2383 ◽  
Author(s):  
Nils Anders Leversen ◽  
Gustavo A. de Souza ◽  
Hiwa Målen ◽  
Swati Prasad ◽  
Inge Jonassen ◽  
...  
Keyword(s):  
Markov Model ◽  
Signal Peptide ◽  
Cleavage Site ◽  
Hidden Markov ◽  
Signal Peptidase ◽  
Secreted Proteins ◽  
Signal Peptides ◽  
Sanger Institute ◽  
Signal Peptidase I ◽  
Peptide Prediction

Secreted proteins play an important part in the pathogenicity of Mycobacterium tuberculosis, and are the primary source of vaccine and diagnostic candidates. A majority of these proteins are exported via the signal peptidase I-dependent pathway, and have a signal peptide that is cleaved off during the secretion process. Sequence similarities within signal peptides have spurred the development of several algorithms for predicting their presence as well as the respective cleavage sites. For proteins exported via this pathway, algorithms exist for eukaryotes, and for Gram-negative and Gram-positive bacteria. However, the unique structure of the mycobacterial membrane raises the question of whether the existing algorithms are suitable for predicting signal peptides within mycobacterial proteins. In this work, we have evaluated the performance of nine signal peptide prediction algorithms on a positive validation set, consisting of 57 proteins with a verified signal peptide and cleavage site, and a negative set, consisting of 61 proteins that have an N-terminal sequence that confirms the annotated translational start site. We found the hidden Markov model of SignalP v3.0 to be the best-performing algorithm for predicting the presence of a signal peptide in mycobacterial proteins. It predicted no false positives or false negatives, and predicted a correct cleavage site for 45 of the 57 proteins in the positive set. Based on these results, we used the hidden Markov model of SignalP v3.0 to analyse the 10 available annotated proteomes of mycobacterial species, including annotations of M. tuberculosis H37Rv from the Wellcome Trust Sanger Institute and the J. Craig Venter Institute (JCVI). When excluding proteins with transmembrane regions among the proteins predicted to harbour a signal peptide, we found between 7.8 and 10.5 % of the proteins in the proteomes to be putative secreted proteins. Interestingly, we observed a consistent difference in the percentage of predicted proteins between the Sanger Institute and JCVI. We have determined the most valuable algorithm for predicting signal peptidase I-processed proteins of M. tuberculosis, and used this algorithm to estimate the number of mycobacterial proteins with the potential to be exported via this pathway.

Evaluating signal peptide prediction methods for Gram-positive bacteria

Biologia ◽  
2009 ◽  
Vol 64 (4) ◽  
Author(s):  
Xiaohui Zhang ◽  
Yudang Li ◽  
Yudong Li
Keyword(s):  
Signal Peptide ◽  
Cleavage Site ◽  
Surface Display ◽  
Extracellular Proteins ◽  
Consensus Method ◽  
Signal Peptides ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Signal Peptide Prediction ◽  
Peptide Prediction

AbstractGram-positive bacteria have been widely investigated for their huge capability to secrete proteins, such as those involved in gene expression, bacterial surface display and bacterial pathogenesis. The N-terminal signal peptide of a secretory protein is responsible for the translocation of polypeptide through the cytoplasmic membrane. Recently, the signal peptide prediction has become a major task in bioinformatics, and many programs with different algorithms were developed to predict signal peptides. In this paper, five prediction programs (SignalP 3.0, PrediSi, Phobius, SOSUIsignal and SIG-Pred) were selected to evaluate their prediction accuracy for signal peptides and cleavage site using 509 unbiased and experimentally verified Gram-positive protein sequences. The results showed that SignalP was the most accurate program in signal peptide (96% accuracy) and cleavage site (83%) prediction. Prediction performance could further be improved by combining multiple methods into consensus prediction, which would increase the accuracy to 98%, and decrease the false positive to zero. When the consensus method was used to predict Bacillus’s extracellular proteins identified by proteomics, more new signal peptides were successfully identified. It could be concluded that the consensus method would be useful to make prediction of signal peptides more reliable.

scholarly journals Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD

2008 ◽  
Vol 191 (21) ◽  
pp. 6732-6740 ◽  
Author(s):  
Sandy Y. M. Ng ◽  
David J. VanDyke ◽  
Bonnie Chaban ◽  
John Wu ◽  
Yoshika Nosaka ◽  
...  
Keyword(s):  
Amino Acids ◽  
Signal Peptide ◽  
Signal Peptidase ◽  
Signal Peptides ◽  
Peptidase Activity ◽  
Vitro Assay ◽  
Type Iv ◽  
Peptide Length ◽  
Short Signal

ABSTRACT In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.

scholarly journals Processing of Human Cytomegalovirus UL37 Mutant Glycoproteins in the Endoplasmic Reticulum Lumen prior to Mitochondrial Importation

2006 ◽  
Vol 80 (14) ◽  
pp. 6771-6783 ◽  
Author(s):  
Manohara S. Mavinakere ◽  
Chad D. Williamson ◽  
Victor S. Goldmacher ◽  
Anamaris M. Colberg-Poley
Keyword(s):  
Endoplasmic Reticulum ◽  
Human Cytomegalovirus ◽  
Cleavage Site ◽  
Secretory Pathway ◽  
Signal Peptidase ◽  
Protein Abundance ◽  
Hydrophobic Core ◽  
Terminal Fragment ◽  
Signal Peptidase I ◽  
Α Helix

ABSTRACT The human cytomegalovirus (HCMV) UL37 glycoprotein (gpUL37) is internally cleaved and its products divergently traffic to mitochondria or are retained in the secretory pathway. To define the requirements for gpUL37 cleavage, residues −1 and −3 of the consensus endoplasmic reticulum (ER) signal peptidase I site within exon 3 (UL37x3) were replaced by bulky tyrosines (gpUL37 cleavage site mutant I). Internal cleavage of this UL37x3 mutant was inhibited, verifying usage of the consensus site at amino acids (aa) 193/194. The full-length mitochondrial species of gpUL37 cleavage site mutant I was N glycosylated and endoglycosidase H sensitive, indicating that ER translocation and processing took place prior to its mitochondrial importation. Moreover, these results suggest that internal cleavage of gpUL37 is not necessary for its N glycosylation. Partial deletion or disruption of the UL37 hydrophobic core immediately upstream of the cleavage site resulted in decreased protein abundance, suggesting that the UL37x3 hydrophobic α-helix contributes to either correct folding or stability of gpUL37. Insertion of the UL37x3 hydrophobic core and cleavage site into pUL37M, a splice variant of gpUL37 which lacks these sequences and is neither proteolytically cleaved nor N glycosylated, resulted in its internal cleavage and N glycosylation. Its NH2-terminal fragment, pUL37M-NH2, was detected more abundantly in mitochondria, while its N-glycosylated C-terminal fragment, gpUL37M-COOH, was detected predominantly in the ER in a manner analogous to that of gpUL37 cleavage products. These results indicate that UL37x3 aa 178 to 205 are prerequisite for gpUL37 internal cleavage and alter UL37 protein topology allowing N glycosylation of its C-terminal sequences. In contrast, the NH2-terminal UL37x1 hydrophobic leader, present in pUL37x1, pUL37M, and gpUL37, is not cleaved from mature UL37 protein, retaining a membrane anchor for UL37 isoforms during trafficking. Taken together, these results suggest that HCMV gpUL37 undergoes sequential trafficking, during which it is ER translocated, processed, and then mitochondrially imported.

scholarly journals A Type I Signal Peptidase Is Required for Pilus Assembly in the Gram-Positive, Biofilm-Forming Bacterium Actinomycesoris

2016 ◽  
Vol 198 (15) ◽  
pp. 2064-2073 ◽  
Author(s):  
Sara D. Siegel ◽  
Chenggang Wu ◽  
Hung Ton-That
Keyword(s):  
Experimental Model ◽  
Signal Peptide ◽  
Biofilm Formation ◽  
Signal Peptidase ◽  
Type I ◽  
Signal Peptides ◽  
Gram Positive ◽  
Content Type ◽  
Lpxtg Motif ◽  
Gram Positive Bacteria

ABSTRACTThe Gram-positive bacteriumActinomycesoris, a key colonizer in the development of oral biofilms, contains 18 LPXTG motif-containing proteins, including fimbrillins that constitute two fimbrial types critical for adherence, biofilm formation, and polymicrobial interactions. Export of these protein precursors, which harbor a signal peptide, is thought to be mediated by the Sec machine and require cleavage of the signal peptide by type I signal peptidases (SPases). Like many Gram-positive bacteria,A. orisexpresses two SPases, named LepB1 and LepB2. The latter has been linked to suppression of lethal “glyco-stress,” caused by membrane accumulation of the LPXTG motif-containing glycoprotein GspA when the housekeeping sortasesrtAis genetically disrupted. Consistent with this finding, we show here that a mutant lackinglepB2andsrtAwas unable to produce high levels of glycosylated GspA and hence was viable. However, deletion of neitherlepB1norlepB2abrogated the signal peptide cleavage and glycosylation of GspA, indicating redundancy of SPases for GspA. In contrast, thelepB2deletion mutant failed to assemble the wild-type levels of type 1 and 2 fimbriae, which are built by the shaft fimbrillins FimP and FimA, respectively; this phenotype was attributed to aberrant cleavage of the fimbrillin signal peptides. Furthermore, thelepB2mutants, including the catalytically inactive S101A and K169A variants, exhibited significant defects in polymicrobial interactions and biofilm formation. Conversely,lepB1was dispensable for the aforementioned processes. These results support the idea that LepB2 is specifically utilized for processing of fimbrial proteins, thus providing an experimental model with which to study the basis of type I SPase specificity.IMPORTANCESec-mediated translocation of bacterial protein precursors across the cytoplasmic membrane involves cleavage of their signal peptide by a signal peptidase (SPase). Like many Gram-positive bacteria,A. orisexpresses two SPases, LepB1 and LepB2. The latter is a genetic suppressor of lethal “glyco-stress” caused by membrane accumulation of glycosylated GspA when the housekeeping sortasesrtAis genetically disrupted. We show here that LepB1 and LepB2 are capable of processing GspA, whereas only LepB2 is required for cleavage of fimbrial signal peptides. This is the first example of a type I SPase dedicated to LPXTG motif-containing fimbrial proteins. Thus,A. orisprovides an experimental model with which to investigate the specificity mechanism of type I SPases.

scholarly journals Comparison of Current Methods for Signal Peptide Prediction in Phytoplasmas

2021 ◽  
Vol 12 ◽  
Author(s):  
Christophe Garcion ◽  
Laure Béven ◽  
Xavier Foissac
Keyword(s):  
Signal Peptide ◽  
Transmembrane Domain ◽  
Signal Peptides ◽  
Prediction Ability ◽  
Signal Peptide Prediction ◽  
General Evaluation ◽  
Increased Sensitivity ◽  
Transmembrane Regions ◽  
Peptide Prediction

Although phytoplasma studies are still hampered by the lack of axenic cultivation methods, the availability of genome sequences allowed dramatic advances in the characterization of the virulence mechanisms deployed by phytoplasmas, and highlighted the detection of signal peptides as a crucial step to identify effectors secreted by phytoplasmas. However, various signal peptide prediction methods have been used to mine phytoplasma genomes, and no general evaluation of these methods is available so far for phytoplasma sequences. In this work, we compared the prediction performance of SignalP versions 3.0, 4.0, 4.1, 5.0 and Phobius on several sequence datasets originating from all deposited phytoplasma sequences. SignalP 4.1 with specific parameters showed the most exhaustive and consistent prediction ability. However, the configuration of SignalP 4.1 for increased sensitivity induced a much higher rate of false positives on transmembrane domains located at N-terminus. Moreover, sensitive signal peptide predictions could similarly be achieved by the transmembrane domain prediction ability of TMHMM and Phobius, due to the relatedness between signal peptides and transmembrane regions. Beyond the results presented herein, the datasets assembled in this study form a valuable benchmark to compare and evaluate signal peptide predictors in a field where experimental evidence of secretion is scarce. Additionally, this study illustrates the utility of comparative genomics to strengthen confidence in bioinformatic predictions.

scholarly journals Efficacy of signal peptide predictors in identifying signal peptides in the experimental secretome of Picrophilous torridus, a thermoacidophilic archaeon

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0255826
Author(s):  
Neelja Singhal ◽  
Anjali Garg ◽  
Nirpendra Singh ◽  
Pallavi Gulati ◽  
Manish Kumar ◽  
...  
Keyword(s):  
Gene Ontology ◽  
Signal Peptide ◽  
Carbon Metabolism ◽  
Metabolic Pathways ◽  
Protein Translocation ◽  
Mass Spectrometric ◽  
Secreted Proteins ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Microbial Adaptation

Secretory proteins are important for microbial adaptation and survival in a particular environment. Till date, experimental secretomes have been reported for a few archaea. In this study, we have identified the experimental secretome of Picrophilous torridus and evaluated the efficacy of various signal peptide predictors (SPPs) in identifying signal peptides (SPs) in its experimental secretome. Liquid chromatography mass spectrometric (LC MS) analysis was performed for three independent P. torridus secretome samples and only those proteins which were common in the three experiments were selected for further analysis. Thus, 30 proteins were finally included in this study. Of these, 10 proteins were identified as hypothetical/uncharacterized proteins. Gene Ontology, KEGG and STRING analyses revealed that majority of the sercreted proteins and/or their interacting partners were involved in different metabolic pathways. Also, a few proteins like malate dehydrogenase (Q6L0C3) were multi-functional involved in different metabolic pathways like carbon metabolism, microbial metabolism in diverse environments, biosynthesis of antibiotics, etc. Multi-functionality of the secreted proteins reflects an important aspect of thermoacidophilic adaptation of P. torridus which has the smallest genome (1.5 Mbp) among nonparasitic aerobic microbes. SPPs like, PRED-SIGNAL, SignalP 5.0, PRED-TAT and LipoP 1.0 identified SPs in only a few secreted proteins. This suggests that either these SPPs were insufficient, or N-terminal SPs were absent in majority of the secreted proteins, or there might be alternative mechanisms of protein translocation in P. torridus.

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