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Cellular and Molecular Life Sciences :... May 2018Proteins routed to the secretory pathway start their journey by being transported across biological membranes, such as the endoplasmic reticulum. The essential nature of... (Review)
Review
Proteins routed to the secretory pathway start their journey by being transported across biological membranes, such as the endoplasmic reticulum. The essential nature of this protein translocation process has led to the evolution of several factors that specifically target the translocon and block translocation. In this review, various translocation pathways are discussed together with known inhibitors of translocation. Properties of signal peptide-specific systems are highlighted for the development of new therapeutic and antimicrobial applications, as compounds can target signal peptides from either host cells or pathogens and thereby selectively prevent translocation of those specific proteins. Broad inhibition of translocation is also an interesting target for the development of new anticancer drugs because cancer cells heavily depend on efficient protein translocation into the endoplasmic reticulum to support their fast growth.
Topics: Animals; Anti-Infective Agents; Antineoplastic Agents; Drug Discovery; Endoplasmic Reticulum; Humans; Models, Molecular; Molecular Targeted Therapy; Neoplasms; Protein Sorting Signals; Protein Transport
PubMed: 29305616
DOI: 10.1007/s00018-017-2743-2 -
Biochimica Et Biophysica Acta Aug 2014Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single... (Review)
Review
Bacterial lipoproteins are peripherally anchored membrane proteins that play a variety of roles in bacterial physiology and virulence in monoderm (single membrane-enveloped, e.g., gram-positive) and diderm (double membrane-enveloped, e.g., gram-negative) bacteria. After export of prolipoproteins through the cytoplasmic membrane, which occurs predominantly but not exclusively via the general secretory or Sec pathway, the proteins are lipid-modified at the cytoplasmic membrane in a multistep process that involves sequential modification of a cysteine residue and cleavage of the signal peptide by the signal II peptidase Lsp. In both monoderms and diderms, signal peptide processing is preceded by acylation with a diacylglycerol through preprolipoprotein diacylglycerol transferase (Lgt). In diderms but also some monoderms, lipoproteins are further modified with a third acyl chain through lipoprotein N-acyl transferase (Lnt). Fully modified lipoproteins that are destined to be anchored in the inner leaflet of the outer membrane (OM) are selected, transported and inserted by the Lol (lipoprotein outer membrane localization) pathway machinery, which consists of the inner-membrane (IM) ABC transporter-like LolCDE complex, the periplasmic LolA chaperone and the OM LolB lipoprotein receptor. Retention of lipoproteins in the cytoplasmic membrane results from Lol avoidance signals that were originally described as the "+2 rule". Surface localization of lipoproteins in diderms is rare in most bacteria, with the exception of several spirochetal species. Type 2 (T2SS) and type 5 (T5SS) secretion systems are involved in secretion of specific surface lipoproteins of γ-proteobacteria. In the model spirochete Borrelia burgdorferi, surface lipoprotein secretion does not follow established sorting rules, but remains dependent on N-terminal peptide sequences. Secretion through the outer membrane requires maintenance of lipoproteins in a translocation-competent unfolded conformation, likely through interaction with a periplasmic holding chaperone, which delivers the proteins to an outer membrane lipoprotein flippase. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Topics: Bacteria; Bacterial Outer Membrane Proteins; Cell Membrane; Lipoproteins; Molecular Chaperones; Periplasm; Periplasmic Binding Proteins; Protein Processing, Post-Translational; Protein Sorting Signals
PubMed: 24780125
DOI: 10.1016/j.bbamcr.2014.04.022 -
The Journal of Biological Chemistry Jul 2008Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic alpha-helixes and beta-sheets....
Lipoproteins consist of lipids solubilized by apolipoproteins. The lipid-binding structural motifs of apolipoproteins include amphipathic alpha-helixes and beta-sheets. Plasma apolipoprotein (apo) M lacks an external amphipathic motif but, nevertheless, is exclusively associated with lipoproteins (mainly high density lipoprotein). Uniquely, however, apoM is secreted to plasma without cleavage of its hydrophobic NH(2)-terminal signal peptide. To test whether the signal peptide serves as a lipoprotein anchor for apoM in plasma, we generated mice expressing a mutated apoM(Q22A) cDNA in the liver (apoM(Q22A)-Tg mice (transgenic mice)) and compared them with mice expressing wild-type human apoM (apoM-Tg mice). The substitution of the amino acid glutamine 22 with alanine in apoM(Q22A) results in secretion of human apoM without a signal peptide. The human apoM mRNA level in liver and the amount of human apoM protein secretion from hepatocytes were similar in apoM-Tg and apoM(Q22A)-Tg mice. Nevertheless, human apoM was not detectable in plasma of apoM(Q22A)-Tg mice, whereas it was easily measured in the apoM-Tg mice. To examine the plasma metabolism, recombinant apoM lacking the signal peptide was produced in Escherichia coli and injected into wild-type mice. The apoM without signal peptide did not associate with lipoproteins and was rapidly cleared in the kidney. Accordingly, ligation of the kidney arteries in apoM(Q22A)-Tg mice resulted in rapid accumulation of human apoM in plasma. The data suggest that hydrophobic signal peptide sequences, if preserved upon secretion, can anchor plasma proteins in lipoproteins. In the case of apoM, this mechanism prevents rapid loss by filtration in the kidney.
Topics: Amino Acid Motifs; Amino Acid Substitution; Animals; Apolipoproteins; Apolipoproteins M; Hepatocytes; Humans; Hydrophobic and Hydrophilic Interactions; Kidney; Lipocalins; Lipoproteins, HDL; Liver; Mice; Mice, Transgenic; Protein Sorting Signals
PubMed: 18460466
DOI: 10.1074/jbc.M800695200 -
Applied and Environmental Microbiology May 2022Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low...
Pichia pastoris is widely used for the production of valuable recombinant proteins. An advantage of P. pastoris over other expression systems is that it secretes low levels of endogenous proteins, which facilitates the purification processes if the desired recombinant proteins are efficiently secreted into the culture medium. However, not all recombinant proteins can be successfully secreted by P. pastoris, especially enzymes that are located in intracellular compartments in their native hosts. Few studies have reported strategies for releasing recombinant proteins which cannot be secreted by standard protocols. Here, we investigated whether this challenge can be addressed using novel secretion leaders. Analysis of the secretome and transcriptome of P. pastoris indicated that the four genes with the highest protein-to-transcript ratios were , , , and , suggesting that their gene products contain efficient secretion leaders. Our data revealed that the signal peptide derived from the gene product conferred secretion competence to certain industrial enzymes, e.g., a nitrilase of Alcaligenes faecalis ZJUTB10, a ribosylnicotinamide kinase of P. pastoris, and a glucose dehydrogenase of Exiguobacterium sibiricum. Therefore, the signal peptide derived from the gene product represents a novel secretion sequence for the secretory expression of recombinant enzymes in P. pastoris. Although P. pastoris is widely used for the secretory production of pharmaceutical proteins, its successful applications in the secretory production of industrial enzymes are limited. The α-mating factor pre-pro leader is the most widely used secretion signal in P. pastoris, but numerous industrial enzymes cannot be secreted using it. The importance of this study is that we identified a signal peptide derived from the gene product which conferred secretion competence to three-quarters of the enzymes tested. This signal peptide derived from the gene product may facilitate the application of P. pastoris in industrial biocatalysis.
Topics: Pichia; Protein Sorting Signals; Recombinant Proteins; Saccharomycetales
PubMed: 35435711
DOI: 10.1128/aem.00296-22 -
Journal of Bacteriology Mar 1991In previous investigations, we have examined the effect of OmpA signal peptide mutations on the secretion of the two heterologous proteins TEM beta-lactamase and...
In previous investigations, we have examined the effect of OmpA signal peptide mutations on the secretion of the two heterologous proteins TEM beta-lactamase and nuclease A. During these studies, we observed that a given signal peptide mutation could affect differentially the processing of precursor OmpA-nuclease or precursor OmpA-lactamase. This observation led us to further investigate the influence of the mature region of a precursor protein on protein export. Preexisting OmpA signal peptide mutations of known secretion phenotype when directing heterologous protein export (nuclease A or beta-lactamase) were fused to the homologous mature OmpA protein. Four signal peptide mutations that have previously been shown to prevent export of nuclease A and beta-lactamase were found to support OmpA protein export, albeit at reduced rates. This remarkable retention of export activity by severely defective precursor OmpA signal peptide mutants may be due to the ability of mature OmpA to interact with the cytoplasmic membrane. In addition, these same signal peptide mutations can affect the level of OmpA synthesis as well as its proper assembly in the outer membrane of Escherichia coli. Two signal peptide mutations dramatically stimulate the rate of precursor OmpA synthesis three- to fivefold above the level observed when a wild-type signal peptide is directing export. The complete removal of the OmpA signal peptide does not result in increased OmpA synthesis. This finding suggests that the signal peptide mutations function positively to stimulate OmpA synthesis, rather than bypass a down-regulatory mechanism effected by a wild-type signal peptide. Overproduction of wild-type precursor OmpA or precursors containing signal peptide mutations which lead to relatively minor kinetic processing defects results in accumulation of an improperly assembled OmpA species (imp-OmpA). In contrast, signal peptide mutations which cause relatively severe processing defects accumulate no or only small quantities of imp-OmpA. All mutations result in equivalent levels of properly assembled OmpA. Thus, a strong correlation between imp-OmpA accumulation and cell toxicity was observed. A mutation in the mature region of OmpA which prevents the proper outer membrane assembly of OmpA was suppressed when export was directed by a severely defective signal peptide. These findings suggest that signal peptide mutations indirectly influence OmpA assembly in the outer membrane by altering both the level and rate of OmpA secretion across the cytoplasmic membrane.
Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; DNA Mutational Analysis; Hot Temperature; Macromolecular Substances; Molecular Sequence Data; Molecular Weight; Mutation; Protein Processing, Post-Translational; Protein Sorting Signals; Structure-Activity Relationship
PubMed: 2002002
DOI: 10.1128/jb.173.6.1997-2005.1991 -
Bioinformatics (Oxford, England) May 2018The identification of signal peptides in protein sequences is an important step toward protein localization and function characterization.
MOTIVATION
The identification of signal peptides in protein sequences is an important step toward protein localization and function characterization.
RESULTS
Here, we present DeepSig, an improved approach for signal peptide detection and cleavage-site prediction based on deep learning methods. Comparative benchmarks performed on an updated independent dataset of proteins show that DeepSig is the current best performing method, scoring better than other available state-of-the-art approaches on both signal peptide detection and precise cleavage-site identification.
AVAILABILITY AND IMPLEMENTATION
DeepSig is available as both standalone program and web server at https://deepsig.biocomp.unibo.it. All datasets used in this study can be obtained from the same website.
CONTACT
SUPPLEMENTARY INFORMATION
Supplementary data are available at Bioinformatics online.
Topics: Bacteria; Deep Learning; Eukaryota; Internet; Protein Sorting Signals; Protein Transport; Sequence Analysis, Protein; Software
PubMed: 29280997
DOI: 10.1093/bioinformatics/btx818 -
Advantages of combined transmembrane topology and signal peptide prediction--the Phobius web server.Nucleic Acids Research Jul 2007When using conventional transmembrane topology and signal peptide predictors, such as TMHMM and SignalP, there is a substantial overlap between these two types of...
When using conventional transmembrane topology and signal peptide predictors, such as TMHMM and SignalP, there is a substantial overlap between these two types of predictions. Applying these methods to five complete proteomes, we found that 30-65% of all predicted signal peptides and 25-35% of all predicted transmembrane topologies overlap. This impairs predictions of 5-10% of the proteome, hence this is an important issue in protein annotation. To address this problem, we previously designed a hidden Markov model, Phobius, that combines transmembrane topology and signal peptide predictions. The method makes an optimal choice between transmembrane segments and signal peptides, and also allows constrained and homology-enriched predictions. We here present a web interface (http://phobius.cgb.ki.se and http://phobius.binf.ku.dk) to access Phobius.
Topics: Computational Biology; Computer Simulation; Cytoplasm; Internet; Membrane Proteins; Models, Molecular; Models, Theoretical; Programming Languages; Protein Conformation; Protein Sorting Signals; Protein Structure, Secondary; Proteomics; Software
PubMed: 17483518
DOI: 10.1093/nar/gkm256 -
Malaria Journal Jan 2020Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding...
BACKGROUND
Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding proteins (OBPs), which influence the chemosensory system and behavioural responses, is essential to understanding the population structure and developing effective control measures against this vector. The present study was designed to identify and analyse the obp1 gene in An. maculipennis.
METHODS
Adults of An. maculipennis sensu stricto were collected in Zanjan Province, northwest of Iran, and gDNAs of female mosquitoes were extracted. Fragments of An. maculipennis obp1 (Amacobp1) gene were amplified using degenerate and specific primers, and some of amplicons were selected for sequencing.
RESULTS
Analysis of amplified products identified that the sequence of Amacobp1 gene was 1341 bp long. This gene contains three exons (5', internal, and 3'of 160, 256, and 18 bp, respectively) and encodes 144 amino acids. The sizes of introns I and II in deduced gene are 268 and 358 nucleotides, respectively. The amino acid sequence in the C-terminal of AmacOBP1 is similar to that of major malaria vector Anopheles species. However, its N-terminal has a specific signal peptide with 19 amino acids. This peptide is conserved in different studied populations, and its sequence of amino acids shows the most variation among anopheline species.
CONCLUSIONS
Degenerate primers in this study are suggested for studying obp1 gene in Anopheles species. Amacobp1 gene is proposed as a molecular marker for the detection of intraspecific ecotypes and diagnosis of different species within Maculipennis Group. Moreover, the N-terminal of AmacOBP1 peptide is recommended as a molecular marker to identify the Amacobp1 expression patterns in different chemosensory organs for assessing the molecular mechanisms and developing novel behavioural disturbance agents to control An. maculipennis.
Topics: Amino Acid Sequence; Animals; Anopheles; Base Sequence; DNA; Exons; Female; Introns; Iran; Male; Mosquito Vectors; Phylogeny; Protein Sorting Signals; Receptors, Odorant; Sequence Alignment
PubMed: 31952536
DOI: 10.1186/s12936-019-3058-6 -
Circulation. Genomic and Precision... Apr 2022Fibronectin () is an essential regulator of homodynamic processes and tissue remodeling that have been proposed to contribute to atherosclerosis. Moreover, recent...
BACKGROUND
Fibronectin () is an essential regulator of homodynamic processes and tissue remodeling that have been proposed to contribute to atherosclerosis. Moreover, recent large-scale genome-wide association studies (GWAS) have linked common genetic variants within the gene to coronary artery disease risk.
METHODS
Public databases were analyzed by 2-Sample Mendelian Randomization. Expression constructs encoding short reporter constructs and full-length plasma variants were introduced in various cell models. Secreted and cellular levels were then analyzed and quantified by SDS-PAGE and fluorescence microscopy. Mass spectrometry and glycosylation analyses were performed to probe possible posttranscriptional differences.
RESULTS
Bioinformatic analyses revealed that common coronary artery disease risk single nucleotide polymorphisms in the locus associate with circulating levels of FN1 and that higher FN1 (fibronectin 1) protein levels in plasma are linked to lower coronary artery disease risk. The coronary artery disease-associated locus encompasses a common polymorphism that translates a L15Q variant situated within the FN1 signal peptide. Introduction of FN1 reporter constructs, differing at position 15, revealed differences in secretion, with the FN1 Q15 variant being less well secreted. Moreover, the L15Q polymorphism was found to alter glycosylation in some cell models but not in human plasma.
CONCLUSIONS
In addition to providing novel functional evidence implicating FN1 in cardioprotection, these findings demonstrate that a common variant within a secretion signal peptide regulates protein function.
Topics: Cardiovascular Diseases; Coronary Artery Disease; Fibronectins; Genome-Wide Association Study; Humans; Protein Sorting Signals
PubMed: 35130031
DOI: 10.1161/CIRCGEN.121.003428 -
The FEBS Journal May 2022Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes....
Biogenesis of hepatitis B virus e antigen is driven by translocon-associated protein complex and regulated by conserved cysteine residues within its signal peptide sequence.
Hepatitis B virus uses e antigen (HBe), which is dispensable for virus infectivity, to modulate host immune responses and achieve viral persistence in human hepatocytes. The HBe precursor (p25) is directed to the endoplasmic reticulum (ER), where cleavage of the signal peptide (sp) gives rise to the first processing product, p22. P22 can be retro-translocated back to the cytosol or enter the secretory pathway and undergo a second cleavage event, resulting in secreted p17 (HBe). Here, we report that translocation of p25 to the ER is promoted by translocon-associated protein complex. We have found that p25 is not completely translocated into the ER; a fraction of p25 is phosphorylated and remains in the cytoplasm and nucleus. Within the p25 sp sequence, we have identified three cysteine residues that control the efficiency of sp cleavage and contribute to proper subcellular distribution of the precore pool.
Topics: Calcium-Binding Proteins; Cysteine; Endoplasmic Reticulum; Hepatitis B; Hepatitis B e Antigens; Hepatitis B virus; Humans; Membrane Glycoproteins; Protein Sorting Signals; Receptors, Cytoplasmic and Nuclear; Receptors, Peptide
PubMed: 34839586
DOI: 10.1111/febs.16304