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Biochemical and Biophysical Research... Jun 2021Signal peptides (SPs) consist of short peptide sequences present at the N-terminal of newly synthesizing proteins and act as a zip code for the translocation of the...
Signal peptides (SPs) consist of short peptide sequences present at the N-terminal of newly synthesizing proteins and act as a zip code for the translocation of the proteins to the endoplasmic reticulum (ER). It was thought that the SPs are intracellularly degraded after translocation to the ER; however, recent studies showed cleaved SPs have diverse roles for controlling cell functions in auto- and/or intercellular manners. In addition, it still remains obscure how SP fragments translocate away from the site where they are produced. Extracellular vesicles (EV) are important for intercellular communication and can transport functional molecules to specific cells. In this study, we show that SPs are involved in EV from T-REx AspALP cells that were transfected with a human APP SP-inducible expression vector. There was no difference in the average particle size or particle concentration of EV collected from T-REx AspALP cells and T-REx Mock cells. When the SP content in the EV was examined by mass spectrometry, the C-terminal fragment of APP SP was identified in the exosomes (SEV) of T-REx AspALP cells. In our preparation of SEV fractions, no ER-specific proteins were detected; therefore, SPs may be included in SEV but not in the debris of degraded ER. This is the first indication that SPs are secreted from cells via EV.
Topics: Alkaline Phosphatase; Amyloid beta-Protein Precursor; Clone Cells; Exosomes; GPI-Linked Proteins; Humans; Isoenzymes; Protein Sorting Signals
PubMed: 33964503
DOI: 10.1016/j.bbrc.2021.04.073 -
International Journal of Molecular... Nov 2021Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short... (Review)
Review
Cleavable endoplasmic reticulum (ER) signal peptides (SPs) and other non-cleavable signal sequences target roughly a quarter of the human proteome to the ER. These short peptides, mostly located at the N-termini of proteins, are highly diverse. For most proteins targeted to the ER, it is the interactions between the signal sequences and the various ER targeting and translocation machineries such as the signal recognition particle (SRP), the protein-conducting channel Sec61, and the signal peptidase complex (SPC) that determine the proteins' target location and provide translocation fidelity. In this review, we follow the signal peptide into the ER and discuss the recent insights that structural biology has provided on the governing principles of those interactions.
Topics: Endopeptidases; Endoplasmic Reticulum; Humans; Protein Sorting Signals; Protein Transport; Proteome; SEC Translocation Channels; Signal Recognition Particle
PubMed: 34769302
DOI: 10.3390/ijms222111871 -
International Journal of Molecular... Nov 2022Signal peptide (SP) mutations are an infrequent cause of inherited retinal diseases (IRDs). We report the genes currently associated with an IRD that possess an SP...
Signal peptide (SP) mutations are an infrequent cause of inherited retinal diseases (IRDs). We report the genes currently associated with an IRD that possess an SP sequence and assess the prevalence of these variants in a multi-institutional retrospective review of clinical genetic testing records. The online databases, RetNet and UniProt, were used to determine which IRD genes possess a SP. A multicenter retrospective review was performed to retrieve cases of patients with a confirmed diagnosis of an IRD and a concurrent SP variant. In silico evaluations were performed with MutPred, MutationTaster, and the signal peptide prediction tool, SignalP 6.0. SignalP 6.0 was further used to determine the locations of the three SP regions in each gene: the N-terminal region, hydrophobic core, and C-terminal region. Fifty-six (56) genes currently associated with an IRD possess a SP sequence. Based on the records review, a total of 505 variants were present in the 56 SP-possessing genes. Six (1.18%) of these variants were within the SP sequence and likely associated with the patients' disease based on in silico predictions and clinical correlation. These six SP variants were in the (early-onset retinal dystrophy), (familial exudative vitreoretinopathy) (FEVR), (FEVR), (retinitis pigmentosa), and (X-linked juvenile retinoschisis) genes. It is important to be aware of SP mutations as an exceedingly rare cause of IRDs. Future studies will help refine our understanding of their role in each disease process and assess therapeutic approaches.
Topics: Humans; Protein Sorting Signals; Retinal Diseases; Retinal Dystrophies; Retina; Retinitis Pigmentosa; Genetic Testing; Mutation; Pedigree; DNA Mutational Analysis; Eye Proteins; Membrane Proteins; Nerve Tissue Proteins; Frizzled Receptors
PubMed: 36362148
DOI: 10.3390/ijms232113361 -
Current Opinion in Genetics &... Oct 2019Many functions of eukaryotic cells are compartmentalized within membrane-bound organelles. One or more cis-encoded signals within a polypeptide sequence typically govern... (Review)
Review
Many functions of eukaryotic cells are compartmentalized within membrane-bound organelles. One or more cis-encoded signals within a polypeptide sequence typically govern protein targeting to and within destination organelles. Perhaps unexpectedly, organelle targeting does not occur with high specificity, but instead is characterized by considerable degeneracy and inefficiency. Indeed, the same peptide signals can target proteins to more than one location, randomized sequences can easily direct proteins to organelles, and many enzymes appear to traverse different subcellular settings across eukaryotic phylogeny. We discuss the potential benefits provided by flexibility in organelle targeting, with a special emphasis on horizontally transferred and de novo proteins. Moreover, we consider how these new organelle residents can be protected and maintained before they contribute to the needs of the cell and promote fitness.
Topics: Amino Acid Sequence; Amoeba; Endoplasmic Reticulum; Eukaryota; Evolution, Molecular; Gene Transfer, Horizontal; Mitochondria; Molecular Chaperones; Phylogeny; Protein Sorting Signals; Protein Transport
PubMed: 31476715
DOI: 10.1016/j.gde.2019.07.012 -
BMC Genomics Jan 2022Proteases catalyze the hydrolysis of peptide bonds of proteins, thereby improving dietary protein digestibility, nutrient availability, as well as flavor and texture of...
BACKGROUND
Proteases catalyze the hydrolysis of peptide bonds of proteins, thereby improving dietary protein digestibility, nutrient availability, as well as flavor and texture of fermented food and feed products. The lactobacilli Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) and Pediococcus acidilactici are widely used in food and feed fermentations due to their broad metabolic capabilities and safe use. However, extracellular protease activity in these two species is low. Here, we optimized protease expression and secretion in L. plantarum and P. acidilactici via a genetic engineering strategy.
RESULTS
To this end, we first developed a versatile and stable plasmid, pUC256E, which can propagate in both L. plantarum and P. acidilactici. We then confirmed expression and secretion of protease PepG1 as a functional enzyme in both strains with the aid of the previously described L. plantarum-derived signal peptide LP_0373. To further increase secretion of PepG1, we carried out a genome-wide experimental screening of signal peptide functionality. A total of 155 predicted signal peptides originating from L. plantarum and 110 predicted signal peptides from P. acidilactici were expressed and screened for extracellular proteolytic activity in the two different strains, respectively. We identified 12 L. plantarum signal peptides and eight P. acidilactici signal peptides that resulted in improved yield of secreted PepG1. No significant correlation was found between signal peptide sequence properties and its performance with PepG1.
CONCLUSION
The vector developed here provides a powerful tool for rapid experimental screening of signal peptides in both L. plantarum and P. acidilactici. Moreover, the set of novel signal peptides identified was widely distributed across strains of the same species and even across some closely related species. This indicates their potential applicability also for the secretion of other proteins of interest in other L. plantarum or P. acidilactici host strains. Our findings demonstrate that screening a library of homologous signal peptides is an attractive strategy to identify the optimal signal peptide for the target protein, resulting in improved protein export.
Topics: High-Throughput Screening Assays; Lactobacillus plantarum; Pediococcus; Pediococcus acidilactici; Peptide Hydrolases; Plasmids; Protein Sorting Signals
PubMed: 35021997
DOI: 10.1186/s12864-022-08292-3 -
Sub-cellular Biochemistry 2018In a compartmentalized cell, correct protein localization is crucial for function of virtually all cellular processes. From the cytoplasm as a starting point, proteins... (Review)
Review
In a compartmentalized cell, correct protein localization is crucial for function of virtually all cellular processes. From the cytoplasm as a starting point, proteins are imported into organelles by specific targeting signals. Many proteins, however, act in more than one cellular compartment. In this chapter, we discuss mechanisms by which proteins can be targeted to multiple organelles with a focus on a novel gene regulatory mechanism, functional translational readthrough, that permits multiple targeting of proteins to the peroxisome and other organelles. In mammals, lactate and malate dehydrogenase are the best-characterized enzymes whose targeting is controlled by functional translational readthrough.
Topics: Animals; Cytoplasm; Mammals; Peroxisomes; Protein Biosynthesis; Protein Sorting Signals; Protein Transport
PubMed: 30378024
DOI: 10.1007/978-981-13-2233-4_8 -
Biochimica Et Biophysica Acta. General... Jan 2020Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the...
BACKGROUND
Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route.
METHODS
Products of recombinant galectin expression in P. pastoris were analyzed by haemagglutination, gel filtration and electrophoresis and lectin blotting as well as mass spectrometry on the level of tryptic peptides and purified glycopeptides(s). Density gradient centrifugation and confocal laser scanning microscopy facilitated localization in transfected human and rat cells, proliferation assays determined activity as growth mediator.
RESULTS
Directing galectin-1 to the classical secretory pathway in yeast produces N-glycosylated protein that is active. It cofractionates and -localizes with calnexin in human cells, only Gal-4 is secreted. Presence of N-glycan(s) reduces affinity of cell binding and growth regulation by Gal-1.
CONCLUSIONS
Folding and activity of a galectin are maintained in signal-peptide-directed routing, N-glycosylation occurs. This pathway would deplete cytoplasm and nucleus of galectin, presence of N-glycans appears to interfere with lattice formation.
GENERAL SIGNIFICANCE
Availability of glycosylated galectins facilitates functional assays to contribute to explain why galectins invariably avoid classical routing for export.
Topics: Animals; Biological Transport; Calnexin; Cell Adhesion; Cell Line; Galectin 1; Galectin 4; Glycosylation; Humans; Polysaccharides; Protein Folding; Protein Sorting Signals; Rats; Signal Transduction
PubMed: 31678146
DOI: 10.1016/j.bbagen.2019.129449 -
Molecules (Basel, Switzerland) Oct 2022The COVID-19 pandemic is caused by SARS-CoV-2; the spike protein is a key structural protein that mediates infection of the host by SARS-CoV-2. In this study, we aimed...
The COVID-19 pandemic is caused by SARS-CoV-2; the spike protein is a key structural protein that mediates infection of the host by SARS-CoV-2. In this study, we aimed to evaluate the effects of signal peptide on the secretion and release of SARS-CoV-2 spike protein. Therefore, we constructed a signal peptide deletion mutant and three signal peptide site-directed mutants. The (H) region and (C) region in the signal peptide of L5F-S13I mutant have changed significantly, compared with wild type, L5F and S13I. We demonstrated the effects of signal peptide on the secretion and synthesis of RBD protein, finding that mutation of S13 to I13 on the signal peptide is more conducive to the secretion of RBD protein, which was mainly due to the shift of the signal peptide cleavage site in the mutant S13I. Here, we not only investigated the structure of the N-terminal signal peptide of the SARS-CoV-2 spike protein but also considered possible secretory pathways. We suggest that the development of drugs that target the signal peptide of the SARS-CoV-2 spike protein may have potential to treat COVID-19 in the future.
Topics: Humans; COVID-19; Pandemics; Protein Sorting Signals; SARS-CoV-2; Spike Glycoprotein, Coronavirus
PubMed: 36235223
DOI: 10.3390/molecules27196688 -
Molecular Microbiology Aug 2021Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated...
Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle.
Topics: Cell Cycle; Cell Membrane; Cell Wall; Lipopolysaccharides; Membrane Proteins; Peptidoglycan; Protein Domains; Protein Sorting Signals; Staphylococcal Protein A; Staphylococcus aureus; Teichoic Acids
PubMed: 33949015
DOI: 10.1111/mmi.14734 -
Proceedings of the National Academy of... Mar 2018The HIV-1 envelope protein (Env) of early-replicating viruses encodes several distinct transmission signatures. One such signature involves a reduced number of potential...
The HIV-1 envelope protein (Env) of early-replicating viruses encodes several distinct transmission signatures. One such signature involves a reduced number of potential N-linked glycosylation sites (PNGs). This transmission signature underscores the importance of posttranslational modifications in the fitness of early-replicating isolates. An additional signature in Env involves the overrepresentation of basic amino acid residues at a specific position in the Env signal peptide (SP). In this report, we investigated the potential impact of this SP signature on gp120 glycosylation and antigenicity. Two recombinant gp120s were constructed, one derived from an isolate that lacks this signature and a second from an early-replicating isolate that includes this signature. Chimeric gp120s were also constructed in which the two SPs were swapped between the isolates. All four gp120s were probed with glycan-, structure- and receptor- specific probes in a surface plasmon resonance binding assay. We found that the SP of Env influences qualitative aspects of Env glycosylation that in turn affect the antigenicity of Env in a major way. The SP impacts the affinity of Env for DC-SIGN, a lectin receptor expressed on dendritic cells that is believed to play a role in mucosal transmission. Additionally, affinity for the monoclonal antibodies 17b and A32, which recognize a CD4-induced, open conformation of Env is also altered. These results demonstrate that natural variation in the SP of HIV Env can significantly impact the antigenicity of mature gp120. Thus, the SP is likely subject to antibody-mediated immune pressure.
Topics: Antibodies, Monoclonal; Dendritic Cells; Glycosylation; HIV Envelope Protein gp120; HIV-1; Protein Sorting Signals; Recombinant Fusion Proteins
PubMed: 29463753
DOI: 10.1073/pnas.1722627115