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PloS One 2020Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a...
Unlike closely related GPCRs, protease-activated receptors (PAR1, PAR2, PAR3, and PAR4) have a predicted signal peptide at their N-terminus, which is encoded by a separate exon, suggesting that the signal peptides of PARs may serve an important and unique function, specific for PARs. In this report, we show that the PAR2 signal peptide, when fused to the N-terminus of IgG-Fc, effectively induced IgG-Fc secretion into culture medium, thus behaving like a classical signal peptide. The presence of PAR2 signal peptide has a strong effect on PAR2 cell surface expression, as deletion of the signal peptide (PAR2ΔSP) led to dramatic reduction of the cell surface expression and decreased responses to trypsin or the synthetic peptide ligand (SLIGKV). However, further deletion of the tethered ligand region (SLIGKV) at the N-terminus rescued the cell surface receptor expression and the response to the synthetic peptide ligand, suggesting that the signal peptide of PAR2 may be involved in preventing PAR2 from intracellular protease activation before reaching the cell surface. Supporting this hypothesis, an Arg36Ala mutation on PAR2ΔSP, which disabled the trypsin activation site, increased the receptor cell surface expression and the response to ligand stimulation. Similar effects were observed when PAR2ΔSP expressing cells were treated with protease inhibitors. Our findings indicated that there is a role of the PAR2 signal peptide in preventing the premature activation of PAR2 from intracellular protease cleavage before reaching the cells surface. The same mechanism may also apply to PAR1, PAR3, and PAR4.
Topics: Animals; COS Cells; Chlorocebus aethiops; Endopeptidases; HEK293 Cells; Humans; Mutation, Missense; Protease Inhibitors; Protein Sorting Signals; Receptor, PAR-1; Receptor, PAR-2; Receptors, Cell Surface; Trypsin
PubMed: 32078628
DOI: 10.1371/journal.pone.0222685 -
Microbial Cell Factories Mar 2018The secretion of biotechnologically or pharmaceutically relevant recombinant proteins into the culture supernatant of a bacterial expression host greatly facilitates... (Review)
Review
The secretion of biotechnologically or pharmaceutically relevant recombinant proteins into the culture supernatant of a bacterial expression host greatly facilitates their downstream processing and significantly reduces the production costs. The first step during the secretion of a desired target protein into the growth medium is its transport across the cytoplasmic membrane. In bacteria, two major export pathways, the general secretion or Sec pathway and the twin-arginine translocation or Tat pathway, exist for the transport of proteins across the plasma membrane. The routing into one of these alternative protein export systems requires the fusion of a Sec- or Tat-specific signal peptide to the amino-terminal end of the desired target protein. Since signal peptides, besides being required for the targeting to and membrane translocation by the respective protein translocases, also have additional influences on the biosynthesis, the folding kinetics, and the stability of the respective target proteins, it is not possible so far to predict in advance which signal peptide will perform best in the context of a given target protein and a given bacterial expression host. As outlined in this review, the most promising way to find the optimal signal peptide for a desired protein is to screen the largest possible diversity of signal peptides, either generated by signal peptide variation using large signal peptide libraries or, alternatively, by optimization of a given signal peptide using site-directed or random mutagenesis strategies.
Topics: Bacterial Proteins; Bacterial Secretion Systems; Escherichia coli; Escherichia coli Proteins; Gram-Positive Bacteria; Industrial Microbiology; Membrane Transport Proteins; Protein Sorting Signals; Protein Transport; Recombinant Proteins; SEC Translocation Channels; Twin-Arginine-Translocation System
PubMed: 29598818
DOI: 10.1186/s12934-018-0901-3 -
Proceedings of the National Academy of... Mar 2024Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established...
Sponges (Porifera) contain many peptide-specialized metabolites with potent biological activities and significant roles in shaping marine ecology. It is well established that symbiotic bacteria produce bioactive "sponge" peptides, both on the ribosome (RiPPs) and nonribosomally. Here, we demonstrate that sponges themselves also produce many bioactive macrocyclic peptides, such as phakellistatins and related proline-rich macrocyclic peptides (PRMPs). Using the sponge transcriptome, methods were developed to find sequences encoding 46 distinct RiPP-type core peptides, of which ten encoded previously identified PRMP sequences. With this basis set, the genome and transcriptome of the sponge was interrogated to find 35 PRMP precursor peptides encoding 31 unique core peptide sequences. At least 11 of these produced cyclic peptides that were present in the sponge and could be characterized by mass spectrometry, including stylissamides A-D and seven previously undescribed compounds. Precursor peptides were encoded in the genome, confirming their animal origin. The peptides contained signal peptide sequences and highly repetitive recognition sequence-core peptide elements with up to 25 PRMP copies in a single precursor. In comparison to sponges without PRMPs, PRMP sponges are incredibly enriched in potentially secreted polypeptides, with >23,000 individual signal peptide encoding genes found in a single transcriptome. The similarities between PRMP biosynthetic genes and neuropeptides in terms of their biosynthetic logic suggest a fundamental biology linked to circular peptides, possibly indicating a widespread and underappreciated diversity of signaling peptide post-translational modifications across the animal kingdom.
Topics: Animals; Peptides; Peptides, Cyclic; Amino Acid Sequence; Bandages; Protein Sorting Signals
PubMed: 38442178
DOI: 10.1073/pnas.2314383121 -
ACS Synthetic Biology Aug 2023Expression of recombinant proteins in mammalian cell factories relies on synthetic assemblies of genetic parts to optimally control flux through the product biosynthetic...
Expression of recombinant proteins in mammalian cell factories relies on synthetic assemblies of genetic parts to optimally control flux through the product biosynthetic pathway. In comparison to other genetic part-types, there is a relative paucity of characterized signal peptide components, particularly for mammalian cell contexts. In this study, we describe a toolkit of signal peptide elements, created using bioinformatics-led and synthetic design approaches, that can be utilized to enhance production of biopharmaceutical proteins in Chinese hamster ovary cell factories. We demonstrate, for the first time in a mammalian cell context, that machine learning can be used to predict how discrete signal peptide elements will perform when utilized to drive endoplasmic reticulum (ER) translocation of specific single chain protein products. For more complex molecular formats, such as multichain monoclonal antibodies, we describe how a combination of in silico and targeted design rule-based in vitro testing can be employed to rapidly identify product-specific signal peptide solutions from minimal screening spaces. The utility of this technology is validated by deriving vector designs that increase product titers ≥1.8×, compared to standard industry systems, for a range of products, including a difficult-to-express monoclonal antibody. The availability of a vastly expanded toolbox of characterized signal peptide parts, combined with streamlined in silico/in vitro testing processes, will permit efficient expression vector re-design to maximize titers of both simple and complex protein products.
Topics: Cricetinae; Animals; Cricetulus; CHO Cells; Protein Sorting Signals; Recombinant Proteins; Antibodies, Monoclonal
PubMed: 37487508
DOI: 10.1021/acssynbio.3c00157 -
Current Atherosclerosis Reports May 2016Plasma high-density lipoprotein cholesterol (HDL-C) concentration is a negative risk factor for atherosclerotic cardiovascular disease (CVD). Despite this, most attempts... (Review)
Review
Plasma high-density lipoprotein cholesterol (HDL-C) concentration is a negative risk factor for atherosclerotic cardiovascular disease (CVD). Despite this, most attempts to raise plasma HDL-C concentrations in a cardioprotective way have failed. Recently, hypotheses about the atheroprotective effects of HDL have shifted away from quantity to quality, mostly HDL function in reverse cholesterol transport. Plasma HDL from CVD patients is a poorer acceptor of cellular cholesterol than plasma from healthy controls, independent of plasma HDL-C concentrations. The function of HDL is likely determined by two other factors, stability and composition. The kinetic instability of HDL, which varies according to subclass, is a likely determinant of its reactivity in response to many HDL-modifying activities. HDL composition is also heterogeneous and variable; all HDL particles contain apo AI but only about two-thirds contain apo AII. This occurs despite the fact that apo AI and apo AII are hepatically secreted on separate HDL that later fuse in plasma. HDL also contains traces of other proteins, some of which have not yet been associated with HDL function. One minor HDL species are those that are secreted with intact signal peptides, which enhances their binding to HDL; these HDL have special properties that are independent of cholesterol transport. Here, we review and provide a perspective about what is currently known about speciated HDL biogenesis in the context of health and disease.
Topics: Animals; Atherosclerosis; Cholesterol; Humans; Lipoproteins, HDL; Protein Sorting Signals
PubMed: 27005803
DOI: 10.1007/s11883-016-0572-7 -
Bioorganic & Medicinal Chemistry Letters May 2020Many current and potential drug targets are membrane-bound or secreted proteins that are expressed and transported via the Sec61 secretory pathway. They are targeted to... (Review)
Review
Many current and potential drug targets are membrane-bound or secreted proteins that are expressed and transported via the Sec61 secretory pathway. They are targeted to translocon channels across the membrane of the endoplasmic reticulum (ER) by signal peptides (SPs), which are temporary structures on the N-termini of their nascent chains. During translation, such proteins enter the lumen and membrane of the ER by a process known as co-translational translocation. Small molecules have been found that interfere with this process, decreasing protein expression by recognizing the unique structures of the SPs of particular proteins. The SP may thus become a validated target for designing drugs for numerous disorders, including certain hereditary diseases.
Topics: Adaptor Proteins, Vesicular Transport; CD4 Antigens; Drug Design; Endoplasmic Reticulum; Humans; Protein Sorting Signals; Protein Transport; Small Molecule Libraries
PubMed: 32209293
DOI: 10.1016/j.bmcl.2020.127115 -
Nature Communications May 2022Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells. We investigate whether engineered microbes can exclusively secrete...
Metabolites are often unable to permeate cell membranes and are thus accumulated inside cells. We investigate whether engineered microbes can exclusively secrete intracellular metabolites because sustainable metabolite secretion holds a great potential for mass-production of high-value chemicals in an efficient and continuous manner. In this study, we demonstrate a synthetic pathway for a metabolite trafficking system that enables lipophilic terpene secretion by yeast cells. When metabolite-binding proteins are tagged with signal peptides, metabolite trafficking is highly achievable; loaded metabolites can be precisely delivered to a desired location within or outside the cell. As a proof of concept, we systematically couple a terpene-binding protein with an export signal peptide and subsequently demonstrate efficient, yet selective terpene secretion by yeast (~225 mg/L for squalene and ~1.6 mg/L for β-carotene). Other carrier proteins can also be readily fused with desired signal peptides, thereby tailoring different metabolite trafficking pathways in different microbes. To the best of our knowledge, this is the most efficient cognate pathway for metabolite secretion by microorganisms.
Topics: Protein Sorting Signals; Saccharomyces cerevisiae; Squalene; Terpenes; beta Carotene
PubMed: 35546160
DOI: 10.1038/s41467-022-30312-9 -
Biochimica Et Biophysica Acta Aug 2014Finding new, effective antibiotics is a challenging research area driven by novel approaches required to tackle unconventional targets. In this review we focus on the... (Review)
Review
Finding new, effective antibiotics is a challenging research area driven by novel approaches required to tackle unconventional targets. In this review we focus on the bacterial protein secretion pathway as a target for eliminating or disarming pathogens. We discuss the latest developments in targeting the Sec-pathway for novel antibiotics focusing on two key components: SecA, the ATP-driven motor protein responsible for driving preproteins across the cytoplasmic membrane and the Type I signal peptidase that is responsible for the removal of the signal peptide allowing the release of the mature protein from the membrane. We take a bird's-eye view of other potential targets in the Sec-pathway as well as other Sec-dependent or Sec-independent protein secretion pathways as targets for the development of novel antibiotics. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Topics: Adenosine Triphosphatases; Anti-Bacterial Agents; Bacteria; Bacterial Proteins; Cell Membrane; Drug Resistance, Bacterial; Humans; Infections; Membrane Proteins; Membrane Transport Proteins; Protein Sorting Signals; Protein Transport; SEC Translocation Channels; SecA Proteins; Secretory Pathway; Serine Endopeptidases
PubMed: 24534745
DOI: 10.1016/j.bbamcr.2014.02.004 -
International Journal of Molecular... Oct 2021Here, we review recent molecular modelling and simulation studies of the Sec translocon, the primary component/channel of protein translocation into the endoplasmic... (Review)
Review
Here, we review recent molecular modelling and simulation studies of the Sec translocon, the primary component/channel of protein translocation into the endoplasmic reticulum (ER) and bacterial periplasm, respectively. Our focus is placed on the eukaryotic Sec61, but we also mention modelling studies on prokaryotic SecY since both systems operate in related ways. Cryo-EM structures are now available for different conformational states of the Sec61 complex, ranging from the idle or closed state over an inhibited state with the inhibitor mycolactone bound near the lateral gate, up to a translocating state with bound substrate peptide in the translocation pore. For all these states, computational studies have addressed the conformational dynamics of the translocon with respect to the pore ring, the plug region, and the lateral gate. Also, molecular simulations are addressing mechanistic issues of insertion into the ER membrane vs. translocation into the ER, how signal-peptides are recognised at all in the translocation pore, and how accessory proteins affect the Sec61 conformation in the co- and post-translational pathways.
Topics: Amino Acid Sequence; Animals; Eukaryota; Eukaryotic Cells; Humans; Models, Molecular; Multiprotein Complexes; Peptides; Protein Binding; Protein Conformation; Protein Sorting Signals; SEC Translocation Channels; Structure-Activity Relationship
PubMed: 34639046
DOI: 10.3390/ijms221910705 -
Biochimica Et Biophysica Acta Aug 2014Type I signal peptidase is the enzyme responsible for cleaving off the amino-terminal signal peptide from proteins that are secreted across the bacterial cytoplasmic... (Review)
Review
Type I signal peptidase is the enzyme responsible for cleaving off the amino-terminal signal peptide from proteins that are secreted across the bacterial cytoplasmic membrane. It is an essential membrane bound enzyme whose serine/lysine catalytic dyad resides on the exo-cytoplasmic surface of the bacterial membrane. This review discusses the progress that has been made in the structural and mechanistic characterization of Escherichia coli type I signal peptidase (SPase I) as well as efforts to develop a novel class of antibiotics based on SPase I inhibition. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Topics: Binding Sites; Cell Membrane; Escherichia coli; Membrane Proteins; Protein Conformation; Protein Sorting Signals; Protein Transport; Serine Endopeptidases
PubMed: 24333859
DOI: 10.1016/j.bbamcr.2013.12.003