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The EMBO Journal Nov 1997Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen... (Comparative Study)
Comparative Study
Secretory proteins and most membrane proteins are synthesized with a signal sequence that is usually cleaved from the nascent polypeptide during transport into the lumen of the endoplasmic reticulum. Using site-specific photo-crosslinking we have followed the fate of the signal sequence of preprolactin in a cell-free system. This signal sequence has an unusually long hydrophilic n-region containing several positively charged amino acid residues. We found that after cleavage by signal peptidase the signal sequence is in contact with lipids and subunits of the signal peptidase complex. The cleaved signal sequence is processed further and an N-terminal fragment is released into the cytosol. This signal peptide fragment was found to interact efficiently with calmodulin. Similar to preprolactin, the signal sequence of the HIV-1 envelope protein p-gp160 has the characteristic feature for calmodulin binding in its n-region. We found that a signal peptide fragment of p-gp160 was released into the cytosol and interacts with calmodulin. Our results suggest that signal peptide fragments of some cellular and viral proteins can interact with cytosolic target molecules. The functional consequences of such interactions remain to be established. However, our data suggest that signal sequences may be functionally more versatile than anticipated up to now.
Topics: Amino Acid Sequence; Calmodulin; Calmodulin-Binding Proteins; Cytosol; HIV Envelope Protein gp160; HIV-1; Membrane Proteins; Membranes; Molecular Sequence Data; Prolactin; Protein Binding; Protein Precursors; Protein Processing, Post-Translational; Protein Sorting Signals; Serine Endopeptidases
PubMed: 9362478
DOI: 10.1093/emboj/16.22.6636 -
Traffic (Copenhagen, Denmark) Feb 2008The relict plastid, or apicoplast, of the malaria parasite Plasmodium falciparum is an essential organelle and a promising drug target. Most apicoplast proteins are... (Review)
Review
The relict plastid, or apicoplast, of the malaria parasite Plasmodium falciparum is an essential organelle and a promising drug target. Most apicoplast proteins are nuclear encoded and post-translationally targeted into the organelle using a bipartite N-terminal extension, consisting of a typical endomembrane signal peptide and a plant-like transit peptide. Apicoplast protein targeting commences through the parasite's secretory pathway. We review recent experimental evidence suggesting that the apicoplast resides in the mainstream endomembrane system proximal to the Golgi. Further, we explore possible mechanisms for translocation of nuclear-encoded apicoplast proteins across the four bounding membranes. Recent insights into the composition of the transit peptide and how it is cleaved and degraded after use are also examined. Characterization of apicoplast targeting has not only shed light on how this group of parasites mediate intracellular protein trafficking events but also it has helped identify new targets for therapeutics. The distinctive leader sequences of apicoplast proteins make them readily identifiable, allowing assembly of a virtual organelle metabolome from the genome. Such analysis has lead to the identification of several biochemical pathways that are absent from the human host and thus represent novel therapeutic targets for parasitic infection.
Topics: Animals; Intracellular Membranes; Membrane Transport Proteins; Models, Biological; Plasmodium falciparum; Plastids; Protein Sorting Signals; Protein Transport
PubMed: 17900270
DOI: 10.1111/j.1600-0854.2007.00660.x -
PloS One 2021Secretory proteins are important for microbial adaptation and survival in a particular environment. Till date, experimental secretomes have been reported for a few...
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.
Topics: Archaea; Chromatography, Liquid; Protein Sorting Signals; Proteome
PubMed: 34358261
DOI: 10.1371/journal.pone.0255826 -
Cells Dec 2022Signal peptides (SPs) and their fragments play important roles as biomarkers and substances with physiological functions in extracellular fluid. We previously reported...
Signal peptides (SPs) and their fragments play important roles as biomarkers and substances with physiological functions in extracellular fluid. We previously reported that SP fragments were released into extracellular fluid via exosomes and bound to calmodulin (CaM), an exosomal component, in a cell-free system. However, it currently remains unclear whether CaM intracellularly interacts with SP fragments or is involved in the trafficking of these fragments to exosomes. Therefore, the present study examined the binding of CaM to SP fragments in T-REx AspALP cells, transformed HEK293 cells expressing amyloid precursor protein (APP) SP flanking a reporter protein, and their exosomes. APP SP fragments were detected in exosomes from T-REx AspALP cells in the absence of W13, a CaM inhibitor, but were present in lower amounts in exosomes from W13-treated cells. Cargo proteins, such as Alix, CD63, and CD81, were increased in W13-treated T-REx AspALP cells but were decreased in their exosomes. Furthermore, CaM interacted with heat shock protein 70 and CD81 in T-REx AspALP cells and this increased in the presence of W13. APP SP fragments were detected in intracellular CaM complexes in the absence of W13, but not in its presence. These results indicate that CaM functions as a key regulator of the transport of SP fragments into exosomes and plays novel roles in the sorting of contents during exosomal biogenesis.
Topics: Humans; Calmodulin; Protein Sorting Signals; HEK293 Cells; Sulfonamides; Amyloid beta-Protein Precursor
PubMed: 36611951
DOI: 10.3390/cells12010158 -
MBio Dec 2017Protein secretion is essential, but how it is managed is poorly understood. In bacteria, most secreted proteins require release from the outer surface of the cytoplasmic...
Protein secretion is essential, but how it is managed is poorly understood. In bacteria, most secreted proteins require release from the outer surface of the cytoplasmic membrane by type I signal peptidase (SPase), which cleaves the mature protein from its membrane-bound N-terminal signal peptide. As the first step that occurs outside the protected cytoplasmic environment and because insufficient activity can rapidly result in the toxic accumulation of preproteins, the activity of SPase is expected to be closely monitored and perhaps supplemented when insufficient. Indeed, we previously demonstrated that inhibition of SPase in results in derepression of the operon, which encodes an alternate mechanism to release proteins. However, in this case, the proteins are released with partially intact signal peptides, with the exception of IsaA, which is released with a virtually intact signal peptide. Here we show that mutation of AyrA [(R233K)] results in constitutive derepression of and that mutation of IsaA's signal peptide [(K2Q)] results in hyperderepression upon SPase inhibition, which also requires AyrA. Further studies demonstrate that the inducing signal for derepression is accumulation of a subset of preproteins with signal peptides that are stable toward further processing and that the signal is critically amplified by the K2Q mutation and relayed to AyrR by AyrA. These results elucidate the mechanism by which monitors and responds to secretion stress. The presence of in other bacteria suggests that it may represent a general strategy linking membrane stress to appropriate transcriptional responses. Bacteria interact with their environment by secreting proteins that perform a myriad of functions, and the final step is the release of the mature protein from the cell surface via the activity of type I signal peptidase (SPase). While the bacterial response to many stresses is understood in some detail, almost nothing is known about how cells respond to secretion stress, such as insufficient SPase activity, which would eventually result in cell death. We previously demonstrated that the inhibition of SPase in results in the derepression of the operon, which can functionally replace SPase, but which is normally repressed by AyrR. We now demonstrate that the inducing signal for derepression is accumulation of a subset of preproteins with signal peptides that are stable to further processing and that the signal is relayed to AyrR via AyrA.
Topics: Bacterial Proteins; Cell Membrane; Gene Expression Regulation, Bacterial; Membrane Proteins; Mutation; Operon; Protein Conformation; Protein Sorting Signals; Protein Transport; Serine Endopeptidases; Staphylococcus aureus; Stress, Physiological; Transcription, Genetic
PubMed: 29233892
DOI: 10.1128/mBio.01507-17 -
Scientific Reports Jul 2019Although diatoms have been extensively studied as bioreactors, only a limited number of efficient gene promoters are available. Therefore, the development of new...
Although diatoms have been extensively studied as bioreactors, only a limited number of efficient gene promoters are available. Therefore, the development of new endogenous promoters is important for the heterologous production of a variety of recombinant proteins. Herein, we identified the most abundant secreted protein in Phaeodactylum tricornutum, designated 'highly abundant secreted protein 1' (HASP1), and characterised the activities of its promoter and signal peptide using green fluorescent protein (GFP) as a reporter. The HASP1 promoter strongly drove GFP expression during all growth phases of P. tricornutum in culture, in contrast to the commonly used fcpA promoter, which is less active during the stationary phase. The HASP1 signal peptide was also sufficient for facilitating efficient secretion of GFP by P. tricornutum. Our findings suggest that both the promoter and the signal peptide of HASP1 can be utilized as novel tools for the overexpression and secretion of recombinant proteins in P. tricornutum.
Topics: Algal Proteins; Diatoms; Green Fluorescent Proteins; Promoter Regions, Genetic; Protein Sorting Signals; Recombinant Fusion Proteins
PubMed: 31289300
DOI: 10.1038/s41598-019-45786-9 -
BMC Bioinformatics Dec 2019Signal peptides play an important role in protein sorting, which is the mechanism whereby proteins are transported to their destination. Recognition of signal peptides...
BACKGROUND
Signal peptides play an important role in protein sorting, which is the mechanism whereby proteins are transported to their destination. Recognition of signal peptides is an important first step in determining the active locations and functions of proteins. Many computational methods have been proposed to facilitate signal peptide recognition. In recent years, the development of deep learning methods has seen significant advances in many research fields. However, most existing models for signal peptide recognition use one-hidden-layer neural networks or hidden Markov models, which are relatively simple in comparison with the deep neural networks that are used in other fields.
RESULTS
This study proposes a convolutional neural network without fully connected layers, which is an important network improvement in computer vision. The proposed network is more complex in comparison with current signal peptide predictors. The experimental results show that the proposed network outperforms current signal peptide predictors on eukaryotic data. This study also demonstrates how model reduction and data augmentation helps the proposed network to predict bacterial data.
CONCLUSIONS
The study makes three contributions to this subject: (a) an accurate signal peptide recognizer is developed, (b) the potential to leverage advanced networks from other fields is demonstrated and (c) important modifications are proposed while adopting complex networks on signal peptide recognition.
Topics: Deep Learning; Neural Networks, Computer; Protein Sorting Signals; Semantics; Software
PubMed: 31861981
DOI: 10.1186/s12859-019-3245-z -
Bioinformatics (Oxford, England) Jun 2023Signal peptides (SPs) are short amino acid segments present at the N-terminus of newly synthesized proteins that facilitate protein translocation into the lumen of the...
MOTIVATION
Signal peptides (SPs) are short amino acid segments present at the N-terminus of newly synthesized proteins that facilitate protein translocation into the lumen of the endoplasmic reticulum, after which they are cleaved off. Specific regions of SPs influence the efficiency of protein translocation, and small changes in their primary structure can abolish protein secretion altogether. The lack of conserved motifs across SPs, sensitivity to mutations, and variability in the length of the peptides make SP prediction a challenging task that has been extensively pursued over the years.
RESULTS
We introduce TSignal, a deep transformer-based neural network architecture that utilizes BERT language models and dot-product attention techniques. TSignal predicts the presence of SPs and the cleavage site between the SP and the translocated mature protein. We use common benchmark datasets and show competitive accuracy in terms of SP presence prediction and state-of-the-art accuracy in terms of cleavage site prediction for most of the SP types and organism groups. We further illustrate that our fully data-driven trained model identifies useful biological information on heterogeneous test sequences.
AVAILABILITY AND IMPLEMENTATION
TSignal is available at: https://github.com/Dumitrescu-Alexandru/TSignal.
Topics: Protein Sorting Signals; Protein Transport; Amino Acids; Benchmarking; Language
PubMed: 37387131
DOI: 10.1093/bioinformatics/btad228 -
MAbs 2022N-terminal heterogeneity resulting from non-uniform signal peptide (SP) cleavage can potentially affect biologics property attributes and result in extended product...
N-terminal heterogeneity resulting from non-uniform signal peptide (SP) cleavage can potentially affect biologics property attributes and result in extended product development timelines. Few studies are available on engineering SPs systematically to address miscleavage issues. Herein, we developed a novel high throughput computational pipeline capable of generating millions of SP mutant sequences that uses the SignalP 5.0 deep learning model to predict which of these mutants are likely to alleviate the N-terminal miscleavage in antibodies. We optimized the parameters to target mutating one or two amino acids at the C-terminus of 84 unique SPs, exhausting all theoretically possible combinations and resulting in a library of 296,077 unique wildtype and mutant signal peptides for in silico screening of each antibody. We applied this method to five antibodies against different targets, with various extent of miscleavage (2.3% to 100%) on their Lambda light chains. In each case, multiple SP mutants were generated, with miscleavage reduced to a non-detectable level and titers comparable with or better than that of the original SPs. Pairwise mutational analysis using an in silico library enriched with high-scoring mutants revealed patterns of amino acids at the C-terminus of SPs, providing insights beyond the "Heijne rule". To our knowledge, no similar approach that combines high throughput in silico mutagenesis and screening with SP cleavage prediction has been reported in the literature. This method can be applied to both the light chain and heavy chain of antibodies, regardless of their initial extent of miscleavage, provides optimized solutions for individual cases, and facilitates the development of antibody therapeutics. Aa, amino acids; CHO, Chinese hamster ovary; CNN, convolutional neural network; CSscore, cleavage site score; CSV, comma-separated values; HC, heavy chain; HEK, human embryonic kidney; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; IGLV, immunoglobulin G Lambda variable; LC, light chain; LCMS, liquid chromatography-mass spectrometry; MS, mass spectrometry; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PEI, polyethylenimine; SP, signal peptide; SPase, signal peptidase; TCEP, tris(2-carboxyethyl) phosphine; TOF, time-of-flight.
Topics: Animals; Antibodies, Monoclonal; CHO Cells; Cricetinae; Cricetulus; Humans; Mutagenesis; Protein Sorting Signals
PubMed: 35275041
DOI: 10.1080/19420862.2022.2044977 -
BMC Biotechnology Nov 2019Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In...
BACKGROUND
Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system.
RESULTS
The constructed integration system comprises of a lactococcal promoter (P or P), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P promoter with USP45 signal peptide which gave very low signals in whole cell ELISA.
CONCLUSION
The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.
Topics: Attachment Sites, Microbiological; Bacteriophages; Genetic Engineering; Genetic Vectors; Genome, Bacterial; Lactococcus lactis; Protein Sorting Signals; Recombination, Genetic; Virus Integration
PubMed: 31775775
DOI: 10.1186/s12896-019-0575-x