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ACS Synthetic Biology Oct 2022DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for...
DsbA leader peptide targets proteins for cotranslational translocation by signal recognition particle (SRP) pathway and has been the standard signal sequence for filamentous phage display of fast-folding Designed Ankyrin Repeat Proteins (DARPins). In contrast, translocation of DARPins via the post-translational pathway, for example, with the commonly used PelB leader, has been reported to be highly inefficient. In this study, two PelB signal sequence libraries were screened covering different regions of the leader peptide for identifying mutants with improved display of DARPins on phage. A PelB variant with the most favorable combination of synonymous mutations in the n-region and hydrophobic substitutions in the h-region increased the display efficiency of a DARPin library 44- and 12-fold compared to PelB and DsbA, respectively. Based on thioredoxin-1 (TrxA) export studies the triple valine mutant PelB DN5 V3 leader was capable of more efficient cotranslational translocation than PelB, but the overall display efficiency improvement over DsbA suggests that besides increased cotranslational translocation other factors contribute to the observed enhancement in DARPin display efficiency.
Topics: Protein Sorting Signals; Signal Recognition Particle; Designed Ankyrin Repeat Proteins; Peptide Library; Hydrophobic and Hydrophilic Interactions; Bacteriophages; Thioredoxins; Codon; Valine
PubMed: 36178799
DOI: 10.1021/acssynbio.2c00260 -
The EMBO Journal Jul 2003The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane... (Review)
Review
The signal recognition particle (SRP) is a ribonucleoprotein particle essential for the targeting of signal peptide-bearing proteins to the prokaryotic plasma membrane or the eukaryotic endoplasmic reticulum membrane for secretion or membrane insertion. SRP binds to the signal peptide emerging from the exit site of the ribosome and forms a ribosome nascent chain (RNC)-SRP complex. The RNC-SRP complex then docks in a GTP-dependent manner with a membrane-anchored SRP receptor and the protein is translocated across or integrated into the membrane through a channel called the translocon. Recently considerable progress has been made in understanding the architecture and function of SRP.
Topics: Amino Acid Sequence; Animals; Endoplasmic Reticulum; Evolution, Molecular; Humans; Membrane Proteins; Models, Biological; Models, Molecular; Protein Conformation; Protein Sorting Signals; Protein Transport; Ribosomes; Sequence Homology, Amino Acid; Signal Recognition Particle
PubMed: 12853463
DOI: 10.1093/emboj/cdg337 -
Nature May 2010Targeting of proteins to appropriate subcellular compartments is a crucial process in all living cells. Secretory and membrane proteins usually contain an amino-terminal...
Targeting of proteins to appropriate subcellular compartments is a crucial process in all living cells. Secretory and membrane proteins usually contain an amino-terminal signal peptide, which is recognized by the signal recognition particle (SRP) when nascent polypeptide chains emerge from the ribosome. The SRP-ribosome nascent chain complex is then targeted through its GTP-dependent interaction with SRP receptor to the protein-conducting channel on endoplasmic reticulum membrane in eukaryotes or plasma membrane in bacteria. A universally conserved component of SRP (refs 1, 2), SRP54 or its bacterial homologue, fifty-four homologue (Ffh), binds the signal peptides, which have a highly divergent sequence divisible into a positively charged n-region, an h-region commonly containing 8-20 hydrophobic residues and a polar c-region. No structure has been reported that exemplifies SRP54 binding of any signal sequence. Here we have produced a fusion protein between Sulfolobus solfataricus SRP54 (Ffh) and a signal peptide connected via a flexible linker. This fusion protein oligomerizes in solution through interaction between the SRP54 and signal peptide moieties belonging to different chains, and it is functional, as demonstrated by its ability to bind SRP RNA and SRP receptor FtsY. We present the crystal structure at 3.5 A resolution of an SRP54-signal peptide complex in the dimer, which reveals how a signal sequence is recognized by SRP54.
Topics: Amino Acid Sequence; Bacterial Proteins; Crystallography, X-Ray; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Multimerization; Protein Sorting Signals; Protein Structure, Quaternary; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; Receptors, Virus; Recombinant Fusion Proteins; Signal Recognition Particle; Structure-Activity Relationship; Sulfolobus solfataricus
PubMed: 20364120
DOI: 10.1038/nature08870 -
MBio Aug 2017The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the...
The general secretory pathway (Sec) and twin-arginine translocase (Tat) operate in parallel to export proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of plant chloroplasts. Substrates are targeted to their respective machineries by N-terminal signal peptides that share a tripartite organization; however, Tat signal peptides harbor a conserved and almost invariant arginine pair that is critical for efficient targeting to the Tat machinery. Tat signal peptides interact with a membrane-bound receptor complex comprised of TatB and TatC components, with TatC containing the twin-arginine recognition site. Here, we isolated suppressors in the signal peptide of the Tat substrate, SufI, that restored Tat transport in the presence of inactivating substitutions in the TatC twin-arginine binding site. These suppressors increased signal peptide hydrophobicity, and copurification experiments indicated that they restored binding to the variant TatBC complex. The hydrophobic suppressors could also act in to suppress substitutions at the signal peptide twin-arginine motif that normally prevent targeting to the Tat pathway. Highly hydrophobic variants of the SufI signal peptide containing four leucine substitutions retained the ability to interact with the Tat system. The hydrophobic signal peptides of two Sec substrates, DsbA and OmpA, containing twin lysine residues, were shown to mediate export by the Tat pathway and to copurify with TatBC. These findings indicate that there is unprecedented overlap between Sec and Tat signal peptides and that neither the signal peptide twin-arginine motif nor the TatC twin-arginine recognition site is an essential mechanistic feature for operation of the Tat pathway. Protein export is an essential process in all prokaryotes. The Sec and Tat export pathways operate in parallel, with the Sec machinery transporting unstructured precursors and the Tat pathway transporting folded proteins. Proteins are targeted to the Tat pathway by N-terminal signal peptides that contain an almost invariant twin-arginine motif. Here, we make the surprising discovery that the twin arginines are not essential for recognition of substrates by the Tat machinery and that this requirement can be bypassed by increasing the signal peptide hydrophobicity. We further show that signal peptides of bona fide Sec substrates can also mediate transport by the Tat pathway. Our findings suggest that key features of the Tat targeting mechanism have evolved to prevent mistargeting of substrates to the Sec pathway rather than being a critical requirement for function of the Tat pathway.
Topics: Adenosine Triphosphatases; Arginine; Bacterial Proteins; Cell Membrane; Escherichia coli Proteins; Hydrophobic and Hydrophilic Interactions; Membrane Transport Proteins; Protein Domains; Protein Sorting Signals; Protein Translocation Systems; Protein Transport; SEC Translocation Channels; SecA Proteins
PubMed: 28765221
DOI: 10.1128/mBio.00909-17 -
International Journal of Molecular... Sep 2022Secretion efficiency of heterologous proteins in the Generally Regarded As Safe (GRAS) is often reported to be insufficiently low due to limitations such as poor...
Secretion efficiency of heterologous proteins in the Generally Regarded As Safe (GRAS) is often reported to be insufficiently low due to limitations such as poor targeting and translocation by the signal peptide or degradation by the host proteases. In this study, the secretion efficiency in the host was enhanced through the utilization of a heterologous signal peptide (SP) SPK1 of . The SPK1 was subjected to site-directed mutations targeting its tripartite N-, H-, and C-domains, and the effect on secretion efficiency as compared to the wild-type SPK1 and native lactococcal USP45 was determined on a reporter nuclease (NUC) of . A Fluorescence Resonance Energy Transfer (FRET) analysis indicated that four out of eight SPK1 variants successfully enhanced the secretion of NUC, with the best mutant, SPKM19, showing elevated secretion efficiency up to 88% (or by 1.4-fold) and an improved secretion activity yield of 0.292 ± 0.122 U/mL (or by 1.7-fold) compared to the wild-type SPK1. Modifications of the SPK1 at the cleavage site C-domain region had successfully augmented the secretion efficiency. Meanwhile, mutations in the H-domain region had resulted in a detrimental effect on the NUC secretion. The development of heterologous SPs with better efficacy than the USP45 has been demonstrated in this study for enhanced secretion of heterologous production and mucosal delivery applications in the lactococcal host.
Topics: Bacterial Proteins; Lactococcus lactis; Mutagenesis, Site-Directed; Protein Sorting Signals; Staphylococcus aureus
PubMed: 36077441
DOI: 10.3390/ijms231710044 -
The Journal of Biological Chemistry May 1987The biological properties of four chemically synthesized signal peptides were compared in mammalian (rabbit reticulocyte) and plant (wheat germ) cell-free protein...
The biological properties of four chemically synthesized signal peptides were compared in mammalian (rabbit reticulocyte) and plant (wheat germ) cell-free protein secretion systems. The precursor-specific region of bovine pre-proparathyroid hormone (preproPTH), [D-Tyr-(+1)]preproPTH-(-29-+1)amide, and a sulfur-free analog, [Nle-(-25), Nle-(-21), Nle-(-18), Ala-(-14), D-Tyr-(+1)]preproPTH-(-29-+1)amide, inhibit the processing of an unrelated precursor protein (pre-prolactin) to its mature secreted form (prolactin) in the mammalian system. In the plant system supplemented with signal recognition particle, the signal peptides arrest translation of both secretory (preprolactin) and cytoplasmic (globin) proteins. One analog, [Nle-(-25), Nle-(-21), Asp-(-18), Ala-(-14), D-Tyr-(+1)]preproPTH-(-29-+1)amide, inhibits preprotein processing in the mammalian system but fails to induce translation arrest in the plant system. A truncated peptide, [N alpha-AcLeu-(-17), Ala-(-14), D-Tyr-(+1)]preproPTH-(-17-+1)amide, lacking the N-terminal (positively charged) region and a portion of the hydrophobic core region, is inactive in both systems. These studies demonstrate that the chemically synthesized signal region of a precursor protein interacts directly with signal recognition particle and functionally mimics the proposed properties of a native signal sequence linked to a nascent protein as it emerges from the ribosome during biosynthesis, and an analog of the signal peptide reveals fundamental differences between the components involved in the protein secretion apparatus in mammals and plants.
Topics: Amino Acid Sequence; Animals; Electrophoresis, Polyacrylamide Gel; Globins; In Vitro Techniques; Mammals; Parathyroid Hormone; Plants; Prolactin; Protein Biosynthesis; Protein Precursors; Protein Sorting Signals; Rabbits; Reticulocytes
PubMed: 3571261
DOI: No ID Found -
BMC Bioinformatics Oct 2005The signal peptide plays an important role in protein targeting and protein translocation in both prokaryotic and eukaryotic cells. This transient, short peptide...
BACKGROUND
The signal peptide plays an important role in protein targeting and protein translocation in both prokaryotic and eukaryotic cells. This transient, short peptide sequence functions like a postal address on an envelope by targeting proteins for secretion or for transfer to specific organelles for further processing. Understanding how signal peptides function is crucial in predicting where proteins are translocated. To support this understanding, we present SPdb signal peptide database http://proline.bic.nus.edu.sg/spdb, a repository of experimentally determined and computationally predicted signal peptides.
RESULTS
SPdb integrates information from two sources (a) Swiss-Prot protein sequence database which is now part of UniProt and (b) EMBL nucleotide sequence database. The database update is semi-automated with human checking and verification of the data to ensure the correctness of the data stored. The latest release SPdb release 3.2 contains 18,146 entries of which 2,584 entries are experimentally verified signal sequences; the remaining 15,562 entries are either signal sequences that fail to meet our filtering criteria or entries that contain unverified signal sequences.
CONCLUSION
SPdb is a manually curated database constructed to support the understanding and analysis of signal peptides. SPdb tracks the major updates of the two underlying primary databases thereby ensuring that its information remains up-to-date.
Topics: Databases, Nucleic Acid; Internet; Peptides; Protein Sorting Signals; Protein Transport; Sequence Analysis, Protein; Systems Integration; User-Computer Interface; Vocabulary, Controlled
PubMed: 16221310
DOI: 10.1186/1471-2105-6-249 -
Proceedings of the National Academy of... Sep 2009Signal peptides of membrane proteins are cleaved by endoplasmic reticulum-resident signal peptidase, and thus, are not present on mature membrane proteins. Here, we...
Signal peptides of membrane proteins are cleaved by endoplasmic reticulum-resident signal peptidase, and thus, are not present on mature membrane proteins. Here, we report that, contrary to the paradigm, the signal peptide of ruminant CD18, the beta-subunit of beta(2)-integrins, is not cleaved. Intriguingly, the intact signal peptide of CD18 is responsible for the susceptibility of ruminant leukocytes to Mannheimia (Pasteurella) haemolytica leukotoxin (Lkt). Inhibition of Lkt-induced cytolysis of ruminant leukocytes by CD18 peptide analogs revealed that the Lkt-binding site is formed by amino acids 5-17 of CD18, which, surprisingly, comprise most of the signal sequence. Flow cytometric analysis of ruminant leukocytes indicated the presence of the signal peptide on mature CD18 molecules expressed on the cell surface. Analysis of transfectants expressing CD18 containing the FLAG epitope at the putative cleavage site confirmed that the signal peptide of bovine CD18 is not cleaved. Analysis of the signal sequence of CD18 of eight ruminants and five nonruminants revealed that the signal sequence of CD18 of ruminants contains "cleavage-inhibiting" Q, whereas that of nonruminants contains "cleavage-conducive" G at position -5 relative to the cleavage site. Site-directed mutagenesis of Q to G at position -5 of the signal peptide of bovine CD18 resulted in the cleavage of the signal peptide and abrogation of cytolysis of transfectants expressing bovine CD18 carrying the Q(-5)G mutation. We propose that engineering cattle and other ruminants to contain this mutation would provide a novel technology to render them less susceptible to pneumonic pasteurellosis and concomitant economic losses.
Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Blotting, Western; CD18 Antigens; Cell Line; Cloning, Molecular; Exotoxins; Flow Cytometry; Mannheimia haemolytica; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Protein Sorting Signals; Ruminants; Sequence Analysis, DNA; Species Specificity
PubMed: 19706410
DOI: 10.1073/pnas.0906775106 -
The Biochemical Journal Sep 1987Recent advances have led to considerable convergence in ideas of the way topogenic sequences act to translocate proteins across various intracellular membranes (Table... (Review)
Review
Recent advances have led to considerable convergence in ideas of the way topogenic sequences act to translocate proteins across various intracellular membranes (Table 2). Whereas co-translational translocation and processing were previously considered the norm at the endoplasmic reticulum membrane, several instances of post-translational translocation into endoplasmic reticulum microsomes in vitro have now been described. However, it must be noted that post-translational translocation in vitro is much less efficient than when endoplasmic reticulum membranes are present during translation, and it is possible that in the intact cell translocation occurs during translation. Movement of proteins into chloroplasts and mitochondria occurs after translation. When translocation is post-translational, proteins may perhaps traverse the membrane as folded domains, and the conformational effects of topogenic sequences on these domains may be as envisaged in Wickner's 'membrane-trigger hypothesis'. Both signal and transit sequences possess amphipathic structures which are capable of interacting with phospholipid bilayers, and these interactions may disturb the bilayer sufficiently to allow entry of the following domains of protein. There is increasing evidence that GTP is required to bind ribosomes and their associated nascent chains to the endoplasmic reticulum membrane. Precisely how the cell's energy is applied to achieve translocation is not clear, but one possibility at the endoplasmic reticulum is that a GTP-hydrolysing transducing mechanism may exist to couple signal sequence receptor binding to movement of the nascent chain across the membrane. Electrochemical gradients are required for protein movement to the mitochondrial inner membrane and across the bacterial inner membrane. Cytoplasmic factors such as SRP, the secA gene product or a 40 kDa protein (for mitochondrial precursors) may act by binding to topogenic sequences and preventing precursor proteins as they are translated from folding into forms which cannot be translocated. Specificity in the cell may be achieved both by targetting interactions between these cytoplasmic factors and their receptors located in target membranes, and also by specific binding of the topogenic sequences to specific proteins integrated into the target membranes. Possible candidates for the latter are the protein of microsomal membranes that reacts with a photoreactive signal peptide to give a 45 kDa complex (Fig. 1), the secY gene product of the bacterial inner membrane, and receptors on the outer membranes of chloroplasts and mitochondria. Whether these aid translocation as well as recognition is not clear.(ABSTRACT TRUNCATED AT 400 WORDS)
Topics: Animals; Biological Transport; Cell Membrane; Peptides; Protein Biosynthesis; Protein Sorting Signals; Proteins; Structure-Activity Relationship; Subcellular Fractions
PubMed: 3318806
DOI: 10.1042/bj2460249 -
International Journal of Molecular... Dec 2021Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades... (Review)
Review
Looking at the variety of the thousands of different polypeptides that have been focused on in the research on the endoplasmic reticulum from the last five decades taught us one humble lesson: no one size fits all. Cells use an impressive array of components to enable the safe transport of protein cargo from the cytosolic ribosomes to the endoplasmic reticulum. Safety during the transit is warranted by the interplay of cytosolic chaperones, membrane receptors, and protein translocases that together form functional networks and serve as protein targeting and translocation routes. While two targeting routes to the endoplasmic reticulum, SRP (signal recognition particle) and GET (guided entry of tail-anchored proteins), prefer targeting determinants at the N- and C-terminus of the cargo polypeptide, respectively, the recently discovered SND (SRP-independent) route seems to preferentially cater for cargos with non-generic targeting signals that are less hydrophobic or more distant from the termini. With an emphasis on targeting routes and protein translocases, we will discuss those functional networks that drive efficient protein topogenesis and shed light on their redundant and dynamic nature in health and disease.
Topics: Animals; Cytosol; Endoplasmic Reticulum; Humans; Protein Sorting Signals; Protein Transport; Proteins; Signal Recognition Particle
PubMed: 35008565
DOI: 10.3390/ijms23010143