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The EMBO Journal Dec 2022Secretory preproteins of the Sec pathway are targeted post-translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains...
Secretory preproteins of the Sec pathway are targeted post-translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains remain non-folded for translocase recognition/translocation. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or traffic further. We sought the structural basis for delayed mature domain folding and how signal peptides regulate it. We compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural cytoplasmic PpiB twin. Global and local hydrogen-deuterium exchange mass spectrometry showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, at different order and rates. PpiA folding is delayed by less hydrophobic native contacts, frustrated residues and a β-turn in the earliest foldon and by signal peptide-mediated disruption of foldon hierarchy. When selected PpiA residues and/or its signal peptide were grafted onto PpiB, they converted it into a slow folder with enhanced in vivo secretion. These structural adaptations in a secretory protein facilitate trafficking.
Topics: Protein Folding; Protein Sorting Signals; Proteins; Cell Membrane; Hydrophobic and Hydrophilic Interactions
PubMed: 36031863
DOI: 10.15252/embj.2022111344 -
Journal of Thrombosis and Haemostasis :... Nov 2023Inherited protein C deficiency (PCD) caused by mutations in protein C (PC) gene (PROC) increases the risk of thrombosis. Missense mutations in PC's signal peptide and...
BACKGROUND
Inherited protein C deficiency (PCD) caused by mutations in protein C (PC) gene (PROC) increases the risk of thrombosis. Missense mutations in PC's signal peptide and propeptide have been reported in patients with PCD, but their pathogenic mechanisms, except mutations in R42 residue, remain unclear.
OBJECTIVES
To investigate the pathogenic mechanisms of inherited PCD caused by 11 naturally occurring missense mutations in PC's signal peptide and propeptide.
METHODS
Using cell-based assays, we evaluated the impact of these mutations on various aspects such as activities and antigens of secreted PC, intracellular PC expression, subcellular localization of a reporter protein, and propeptide cleavage. Additionally, we investigated their effect on pre-messenger RNA (pre-mRNA) splicing using a minigene splicing assay.
RESULTS
Our data revealed that certain missense mutations (L9P, R32C, R40C, R38W, and R42C) disrupted PC secretion by impeding cotranslational translocation to the endoplasmic reticulum or causing endoplasmic reticulum retention. Additionally, some mutations (R38W and R42L/H/S) resulted in abnormal propeptide cleavage. However, a few missense mutations (Q3P, W14G, and V26M) did not account for PCD. Using a minigene splicing assay, we observed that several variations (c.8A>C, c.76G>A, c.94C>T, and c.112C>T) increased the incidence of aberrant pre-mRNA splicing.
CONCLUSION
Our findings suggest that variations in PC's signal peptide and propeptide have varying effects on the biological process of PC, including posttranscriptional pre-mRNA splicing, translation, and posttranslational processing. Additionally, a variation could affect the biological process of PC at multiple levels. Except for W14G, our results provide a clear understanding of the relationship between PROC genotype and inherited PCD.
Topics: Humans; Protein C Deficiency; RNA Precursors; Protein Sorting Signals; RNA Splicing; Mutation; Mutation, Missense; RNA, Messenger
PubMed: 37393002
DOI: 10.1016/j.jtha.2023.06.021 -
Protein and Peptide Letters 2022Bacillus subtilis is a Gram-positive bacterium that has gained an unprecedented reputation as an expression system at the industrial scale due to characteristics, such... (Review)
Review
Bacillus subtilis is a Gram-positive bacterium that has gained an unprecedented reputation as an expression system at the industrial scale due to characteristics, such as GRAS (Generally recognized as safe), ease of genetic manipulation, high growth rate on the cheap substrate, and short fermentation cycle. This expression system has been widely accepted for the production of various chemicals, pharmaceutical products, food products, proteins, and enzymes. However, there are various hurdles to optimizing the production of heterologous protein in this expression system due to a lack of understanding regarding metabolic pathways and expression elements. In this review, we have emphasized strategies that can enhance the expression level of heterologous proteins in B. subtilis. These strategies include optimization of B. Subtilis strain, expression elements, such as promotors, UTR (Untranslated region), RBS (Ribosome binding site), signal peptide, and metabolic pathways. Finally, contemporary challenges and future perspectives of B. subtilis as an industrial-scale expression system are discussed.
Topics: Bacillus subtilis; Protein Sorting Signals; Promoter Regions, Genetic; Fermentation; Metabolic Networks and Pathways
PubMed: 35927811
DOI: 10.2174/0929866529666220803163335 -
Biochimica Et Biophysica Acta.... Oct 2022Secreted proteins contain an N-terminal signal peptide to guide them through the secretion pathway. Once the protein is translocated, the signal peptide is removed by a... (Review)
Review
Secreted proteins contain an N-terminal signal peptide to guide them through the secretion pathway. Once the protein is translocated, the signal peptide is removed by a signal peptidase, such as signal peptidase I. The signal peptide has been extensively studied and reviewed; however, the mature region has not been the focus of review. Here we cover the experimental evidence that highlights the important role of the mature region amino acid residues in both the efficiency and the ability of secreted proteins to be successfully exported via secretion pathways and cleaved by signal peptidase I.
Topics: Amino Acid Sequence; Bacterial Proteins; Biological Transport; Escherichia coli; Protein Sorting Signals
PubMed: 35798072
DOI: 10.1016/j.bbamem.2022.184000 -
Methods in Molecular Biology (Clifton,... 2017SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the...
SignalP is the currently most widely used program for prediction of signal peptides from amino acid sequences. Proteins with signal peptides are targeted to the secretory pathway, but are not necessarily secreted. After a brief introduction to the biology of signal peptides and the history of signal peptide prediction, this chapter will describe all the options of the current version of SignalP and the details of the output from the program. The chapter includes a case study where the scores of SignalP were used in a novel way to predict the functional effects of amino acid substitutions in signal peptides.
Topics: Algorithms; Computational Biology; Protein Sorting Signals; Proteins; Sequence Analysis, Protein; Software
PubMed: 28451972
DOI: 10.1007/978-1-4939-7015-5_6 -
Database : the Journal of Biological... Feb 2024Detecting changes in the dynamics of secreted proteins in serum has been a challenge for proteomics. Enter secreted protein database (SEPDB), an integrated secretory...
Detecting changes in the dynamics of secreted proteins in serum has been a challenge for proteomics. Enter secreted protein database (SEPDB), an integrated secretory proteomics database offering human, mouse and rat secretory proteomics datasets collected from serum, exosomes and cell culture media. SEPDB compiles secreted protein information from secreted protein database, UniProt and Human Protein Atlas databases to annotate secreted proteomics data based on protein subcellular localization and disease markers. SEPDB integrates the latest predictive modeling techniques to measure deviations in the distribution of signal peptide structures of secreted proteins, extends signal peptide sequence prediction by excluding transmembrane structural domain proteins and updates the validation analysis pipeline for secreted proteins. To establish tissue-specific profiles, we have also created secreted proteomics datasets associated with different human tissues. In addition, we provide information on heterogeneous receptor network organizational relationships, reflective of the complex functional information inherent in the molecular structures of secreted proteins that serve as ligands. Users can take advantage of the Refreshed Search, Analyze, Browse and Download functions of SEPDB, which is available online at https://sysomics.com/SEPDB/. Database URL: https://sysomics.com/SEPDB/.
Topics: Animals; Mice; Rats; Humans; Databases, Protein; Proteins; Proteomics; Protein Sorting Signals
PubMed: 38345567
DOI: 10.1093/database/baae007 -
Medical Mycology Dec 2022To identify the infection mechanism of Aspergillus fumigatus, which is an opportunistic fungal pathogen, we analyzed the expression profile of the whole genome of A....
To identify the infection mechanism of Aspergillus fumigatus, which is an opportunistic fungal pathogen, we analyzed the expression profile of the whole genome of A. fumigatus during the infection of murine macrophages. A previously reported RNA-seq data analysis showed that many genes involved in cell wall synthesis were upregulated during the infection process. Interestingly, AfSec1 (3g12840), which encodes a putative signal peptidase, was upregulated dramatically, and its putative target protein Gel1, which encodes a 1,3-β-glucanosyltransferase, was also upregulated. Instead of the AfSec1 deletion strain, the AfSec1-ΔP strain was constructed, in which the promoter region of AfSec1 was deleted, and AfSec1 expression was not detected in the AfSec1-ΔP strain. The expression of AfSec1 was recovered by the introduction of the promoter region (the AfSec1-ΔP/P strain). The nonprocessed form of Gel1 was identified in the AfSec1-ΔP strain, which lacked the promoter, but mature forms of Gel1 were found in the wild-type and in AfSec1-ΔP/P, which was the promoter complementation strain. In the plate assay, the AfSec1-ΔP strain showed higher sensitivity against caspofungin than the wild-type. However, compared with the wild-type, the deletion strain showed no difference in the sensitivity to other antifungal drugs, such as amphotericin B and voriconazole, which inhibit different targets compared with caspofungin. The AfSec1-ΔP strain exhibited ∼20% lower levels of β-glucan in the cell wall than the wild-type. Finally, the virulence decreased when the promoter region of AfSec1 was deleted, as observed in the murine infection test and conidia-killing assay using human macrophages and neutrophils. These results suggest that AfSec1 exerts signal peptidase activity on its target Gel1 and has an important role in fungal pathogenesis.
Topics: Animals; Mice; Humans; Aspergillus fumigatus; Caspofungin; Fungal Proteins; Protein Sorting Signals; Antifungal Agents
PubMed: 36657388
DOI: 10.1093/mmy/myad005 -
International Journal of Molecular... Mar 2022Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity...
Signal peptides (SPs) not only mediate targeting to the endoplasmic reticulum (ER) but also play important roles as biomarkers and substances with physiological activity in extracellular fluids including blood. SPs are thought to be degraded intracellularly, making it unclear how they are transported from the ER to the extracellular fluid. In a recent study, we showed that a C-terminal fragment of the SP of a type I membrane protein, amyloid precursor protein (APP), was secreted into the extracellular fluid via exosomes using transformed HEK293 cells expressing APP SP flanking a reporter protein. In the present study, we demonstrate that a N-terminal fragment of the SP from a type II membrane protein, human placental secreted alkaline phosphatase (SEAP), is contained in exosomes and secreted into the extracellular fluid using HEK-Blue hTLR3 cells, which express both a human toll-like receptor 3 gene and an inducible SEAP reporter gene. When HEK-Blue hTLR3 cells were stimulated with a TLR3 ligand, a N-terminal fragment of SEAP SP in exosomes was increased in parallel with SEAP secretion in a concentration-dependent manner. These results indicated that SP fragments are exosomal components. In addition, migrating SP fragments were determined by characteristics of the signal-anchor sequence of membrane proteins. Furthermore, we found that SP fragments could bind to calmodulin (CALM), which is a cytosolic protein and also a component of exosomes, suggesting its involvement in the transportation of SP fragments from the endoplasmic reticulum to exosomes.
Topics: Amyloid beta-Protein Precursor; Exosomes; Female; HEK293 Cells; Humans; Peptide Fragments; Placenta; Pregnancy; Protein Sorting Signals
PubMed: 35328557
DOI: 10.3390/ijms23063137 -
Journal of Proteome Research Nov 2015Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop...
Limited proteolysis is a pivotal mechanism regulating protein functions. Identifying physiologically or pathophysiologically relevant cleavage sites helps to develop molecular tools that can be used for diagnostics or therapeutics. During proteolysis of secretory and membrane proteins, part of the cleaved protein is liberated and destined to undergo degradation but should retain original cleavage sites created by proteolytic enzymes. We profiled endogenous peptides accumulated for 4 h in media conditioned by primary cultured rat cardiac fibroblasts. A total of 3916 redundant peptide sequences from 94 secretory proteins and membrane proteins served to identify limited cleavage sites, both annotated and unannotated, for signal peptide or propeptide removal, peptide hormone processing, ectodomain shedding, and regulated intramembrane proteolysis. Incorrectly predicted signal cleavage sites are found in typical proteins such as extracellular matrix proteins and the peptide hormone precursor adrenomedullin ADM. The revealed signal peptide cleavage site for ADM was experimentally verified by identifying the major molecular form of flanking proadrenomedullin N-terminal peptide. We suggest that profiling of endogenous peptides, like transcriptome sequence reads, makes sense in regular cells such as fibroblasts and that peptidomics provides insight into proteolysis-regulated protein functions.
Topics: Adrenomedullin; Amino Acid Sequence; Animals; Cell Membrane; Extracellular Matrix Proteins; Fibroblasts; Membrane Proteins; Molecular Sequence Annotation; Molecular Sequence Data; Myocardium; Peptide Fragments; Primary Cell Culture; Protein Sorting Signals; Protein Structure, Tertiary; Proteolysis; Proteomics; Rats; Transcriptome
PubMed: 26479776
DOI: 10.1021/acs.jproteome.5b00820 -
Plant Science : An International... May 2015Approximately 18% of Arabidopsis thaliana proteins encode a signal peptide for translocation to the endoplasmic reticulum (ER), the gateway of the eukaryotic secretory... (Review)
Review
Approximately 18% of Arabidopsis thaliana proteins encode a signal peptide for translocation to the endoplasmic reticulum (ER), the gateway of the eukaryotic secretory pathway. However, it was recently discovered that some ER proteins can undergo both co-translational import into the ER/secretory pathway and trafficking to compartments outside of the secretory pathway. This phenomenon is observed among members of the protein disulfide isomerase (PDI) family, which are traditionally regarded as ER enzymes involved in protein folding. Although classical PDIs possess an N-terminal signal peptide and a C-terminal ER retention signal, some also dual localize to secretory and non-secretory compartments, including mammalian PDI ERp57, Chlamydomonas reinhardtii PDI RB60, and A. thaliana AtPDI2. ERp57 is present in both the ER and nucleus where it influences gene transcription. RB60 localizes to the ER and chloroplast where it modulates the redox state of polyadenylate-binding protein RB47. AtPDI2, which interacts with transcription factor MEE8, localizes to the ER-secretory pathway and the nucleus. A model proposing secretory trafficking of AtPDI2 and nuclear co-translocation of an AtPDI2-MEE8 complex illustrates the diversity of dual targeting mechanisms, the multifunctional roles of some PDIs, and the potential co-translocation of other proteins to multiple subcellular compartments.
Topics: Arabidopsis; Cell Nucleus; Chlamydomonas reinhardtii; Chloroplasts; Endoplasmic Reticulum; Protein Disulfide-Isomerases; Protein Sorting Signals; Protein Transport; Secretory Pathway
PubMed: 25804820
DOI: 10.1016/j.plantsci.2015.02.013