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Cell Apr 2020Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway...
Many cytosolic proteins lacking a signal peptide, called leaderless cargoes, are secreted through unconventional secretion. Vesicle trafficking is a major pathway involved. It is unclear how leaderless cargoes enter into the vesicle. Here, we find a translocation pathway regulating vesicle entry and secretion of leaderless cargoes. We identify TMED10 as a protein channel for the vesicle entry and secretion of many leaderless cargoes. The interaction of TMED10 C-terminal region with a motif in the cargo accounts for the selective release of the cargoes. In an in vitro reconstitution assay, TMED10 directly mediates the membrane translocation of leaderless cargoes into the liposome, which is dependent on protein unfolding and enhanced by HSP90s. In the cell, TMED10 localizes on the endoplasmic reticulum (ER)-Golgi intermediate compartment and directs the entry of cargoes into this compartment. Furthermore, cargo induces the formation of TMED10 homo-oligomers which may act as a protein channel for cargo translocation.
Topics: Animals; Biological Transport; Cell Line; Cell Line, Tumor; Cell Membrane; Cytosol; Endoplasmic Reticulum; Golgi Apparatus; Humans; Mice; Mice, Inbred C57BL; Protein Sorting Signals; Protein Translocation Systems; Protein Transport; Proteins; Secretory Pathway; Vesicular Transport Proteins
PubMed: 32272059
DOI: 10.1016/j.cell.2020.03.031 -
Natural Product Reports Jan 2021Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in... (Review)
Review
Covering: up to June 2020Ribosomally-synthesized and post-translationally modified peptides (RiPPs) are a large group of natural products. A community-driven review in 2013 described the emerging commonalities in the biosynthesis of RiPPs and the opportunities they offered for bioengineering and genome mining. Since then, the field has seen tremendous advances in understanding of the mechanisms by which nature assembles these compounds, in engineering their biosynthetic machinery for a wide range of applications, and in the discovery of entirely new RiPP families using bioinformatic tools developed specifically for this compound class. The First International Conference on RiPPs was held in 2019, and the meeting participants assembled the current review describing new developments since 2013. The review discusses the new classes of RiPPs that have been discovered, the advances in our understanding of the installation of both primary and secondary post-translational modifications, and the mechanisms by which the enzymes recognize the leader peptides in their substrates. In addition, genome mining tools used for RiPP discovery are discussed as well as various strategies for RiPP engineering. An outlook section presents directions for future research.
Topics: Biological Products; Computational Biology; Enzymes; Hydroxylation; Methylation; Peptides; Phosphorylation; Protein Engineering; Protein Processing, Post-Translational; Protein Sorting Signals; Ribosomes
PubMed: 32935693
DOI: 10.1039/d0np00027b -
Nucleic Acids Research Jul 2022The prediction of protein subcellular localization is of great relevance for proteomics research. Here, we propose an update to the popular tool DeepLoc with...
The prediction of protein subcellular localization is of great relevance for proteomics research. Here, we propose an update to the popular tool DeepLoc with multi-localization prediction and improvements in both performance and interpretability. For training and validation, we curate eukaryotic and human multi-location protein datasets with stringent homology partitioning and enriched with sorting signal information compiled from the literature. We achieve state-of-the-art performance in DeepLoc 2.0 by using a pre-trained protein language model. It has the further advantage that it uses sequence input rather than relying on slower protein profiles. We provide two means of better interpretability: an attention output along the sequence and highly accurate prediction of nine different types of protein sorting signals. We find that the attention output correlates well with the position of sorting signals. The webserver is available at services.healthtech.dtu.dk/service.php?DeepLoc-2.0.
Topics: Humans; Proteins; Protein Sorting Signals; Eukaryota; Protein Transport; Language; Databases, Protein; Computational Biology; Subcellular Fractions
PubMed: 35489069
DOI: 10.1093/nar/gkac278 -
PloS One 2015Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to...
Translocation of a nascent protein from the cytosol into the ER mediated by its signal peptide is a critical step in protein secretion. The aim of this work was to develop a platform technology to optimize the signal peptides for high level production of therapeutic antibodies in CHO cells. A database of signal peptides from a large number of human immunoglobulin (Ig) heavy chain (HC) and kappa light chain (LC) was generated. Most of the HC signal peptides contain 19 amino acids which can be divided into three domains and the LC signal peptides contain 22 amino acids. The signal peptides were then clustered according to sequence similarity. Based on the clustering, 8 HC and 2 LC signal peptides were analyzed for their impacts on the production of 5-top selling antibody therapeutics, namely, Herceptin, Avastin, Remicade, Rituxan, and Humira. The best HC and LC signal peptides for producing these 5 antibodies were identified. The optimized signal peptides for Rituxan is 2-fold better compared to its native signal peptides which are available in the public database. Substitution of a single amino acid in the optimized HC signal peptide for Avastin reduced its production significantly. Mass spectrometry analyses revealed that all optimized signal peptides are accurately removed in the mature antibodies. The results presented in this report are particularly important for the production of these 5 antibodies as biosimilar drugs. They also have the potential to be the best signal peptides for the production of new antibodies in CHO cells.
Topics: Animals; Antibodies, Monoclonal; CHO Cells; Cricetulus; Immunoglobulin G; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Mass Spectrometry; Protein Sorting Signals
PubMed: 25706993
DOI: 10.1371/journal.pone.0116878 -
Journal of Biomedical Science May 2023The SCUBE [Signal peptide-Complement C1r/C1s, Uegf, Bmp1 (CUB)-Epithelial growth factor domain-containing protein] family consists of three proteins in vertebrates,... (Review)
Review
The SCUBE [Signal peptide-Complement C1r/C1s, Uegf, Bmp1 (CUB)-Epithelial growth factor domain-containing protein] family consists of three proteins in vertebrates, SCUBE1, 2 and 3, which are highly conserved in zebrafish, mice and humans. Each SCUBE gene encodes a polypeptide of approximately 1000 amino acids that is organized into five modular domains: (1) an N-terminal signal peptide sequence, (2) nine tandem epidermal growth factor (EGF)-like repeats, (3) a large spacer region, (4) three cysteine-rich (CR) motifs, and (5) a CUB domain at the C-terminus. Murine Scube genes are expressed individually or in combination during the development of various tissues, including those in the central nervous system and the axial skeleton. The cDNAs of human SCUBE orthologs were originally cloned from vascular endothelial cells, but SCUBE expression has also been found in platelets, mammary ductal epithelium and osteoblasts. Both soluble and membrane-associated SCUBEs have been shown to play important roles in physiology and pathology. For instance, upregulation of SCUBEs has been reported in acute myeloid leukemia, breast cancer and lung cancer. In addition, soluble SCUBE1 is released from activated platelets and can be used as a clinical biomarker for acute coronary syndrome and ischemic stroke. Soluble SCUBE2 enhances distal signaling by facilitating the secretion of dual-lipidated hedgehog from nearby ligand-producing cells in a paracrine manner. Interestingly, the spacer regions and CR motifs can increase or enable SCUBE binding to cell surfaces via electrostatic or glycan-lectin interactions. As such, membrane-associated SCUBEs can function as coreceptors that enhance the signaling activity of various serine/threonine kinase or tyrosine kinase receptors. For example, membrane-associated SCUBE3 functions as a coreceptor that promotes signaling in bone morphogenesis. In humans, SCUBE3 mutations are linked to abnormalities in growth and differentiation of both bones and teeth. In addition to studies on human SCUBE function, experimental results from genetically modified mouse models have yielded important insights in the field of systems biology. In this review, we highlight novel molecular discoveries and critical directions for future research on SCUBE proteins in the context of cancer, skeletal disease and cardiovascular disease.
Topics: Humans; Animals; Mice; Zebrafish; Endothelial Cells; Cell Membrane; Protein Sorting Signals; Biology; Calcium-Binding Proteins; Adaptor Proteins, Signal Transducing
PubMed: 37237303
DOI: 10.1186/s12929-023-00925-3 -
Science Advances Apr 2022Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, virulence cassette (PVC) was...
Extracellular contractile injection systems (eCISs) are widespread bacterial nanomachines that resemble T4 phage tail. As a typical eCIS, virulence cassette (PVC) was proposed to inject toxins into eukaryotic cells by puncturing the cell membrane from outside. This makes it an ideal tool for protein delivery in biomedical research. However, how to manipulate this nanocomplex as a molecular syringe is still undetermined. Here, we identify that one group of N-terminal signal peptide (SP) sequences are crucial for the effector loading into the inner tube of PVC complex. By application of genetic operation, cryo-electron microscopy, in vitro translocation assays, and animal experiments, we show that, under the guidance of the SP, numerous prokaryotic and eukaryotic proteins can be loaded into PVC to exert their functions across cell membranes. We therefore might customize PVC as a potent protein delivery nanosyringe for biotherapy by selecting cargo proteins in a broad spectrum, regardless of their species, sizes, and charges.
Topics: Animals; Cryoelectron Microscopy; Photorhabdus; Polyvinyl Chloride; Protein Sorting Signals; Virulence
PubMed: 35486720
DOI: 10.1126/sciadv.abm2343 -
Nature Chemical Biology Sep 2023Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been...
Preventing the biogenesis of disease-relevant proteins is an attractive therapeutic strategy, but attempts to target essential protein biogenesis factors have been hampered by excessive toxicity. Here we describe KZR-8445, a cyclic depsipeptide that targets the Sec61 translocon and selectively disrupts secretory and membrane protein biogenesis in a signal peptide-dependent manner. KZR-8445 potently inhibits the secretion of pro-inflammatory cytokines in primary immune cells and is highly efficacious in a mouse model of rheumatoid arthritis. A cryogenic electron microscopy structure reveals that KZR-8445 occupies the fully opened Se61 lateral gate and blocks access to the lumenal plug domain. KZR-8445 binding stabilizes the lateral gate helices in a manner that traps select signal peptides in the Sec61 channel and prevents their movement into the lipid bilayer. Our results establish a framework for the structure-guided discovery of novel therapeutics that selectively modulate Sec61-mediated protein biogenesis.
Topics: Animals; Mice; Protein Sorting Signals; Protein Transport; Membrane Proteins; SEC Translocation Channels; Protein Biosynthesis
PubMed: 37169961
DOI: 10.1038/s41589-023-01326-1 -
Protein Science : a Publication of the... Dec 2022Signal peptide (SP) plays an important role in membrane targeting for insertion of secretory and membrane proteins during translocation processes in prokaryotes and...
Signal peptide (SP) plays an important role in membrane targeting for insertion of secretory and membrane proteins during translocation processes in prokaryotes and eukaryotes. Beside the targeting functions, SP has also been found to affect the stability and folding of several proteins. Serum amyloid A (SAA) proteins are apolipoproteins responding to acute-phase inflammation. The fibrillization of SAA results in a protein misfolding disease named amyloid A (AA) amyloidosis. The main disease-associated isoform of human SAA, SAA1.1, is expressed as a precursor protein with an N-terminal signal peptide composed of 18 residues. The cleavage of the SP generates mature SAA1.1. To investigate whether the SP affects properties of SAA1.1, we systematically examined the structure, protein stability, and fibrillization propensity of pre-SAA1.1, which possesses the SP, and Ser-SAA1.1 without the SP but containing with an additional N-terminal serine residue. We found that the presence of the SP did not significantly affect the predominant helical structure but changed the tertiary conformation as evidenced by intrinsic fluorescence and exposed hydrophobic surfaces. Pre-SAA1.1 and Ser-SAA1.1 formed distinct oligomeric assemblies in which pre-SAA1.1 populated as tetramer and octamer, whereas Ser-SAA1.1 existed as a predominant hexamer. Pre-SAA1.1 was found significantly more stable than Ser-SAA1.1 upon thermal and chemical unfolding. Ser-SAA1.1, but not pre-SAA1.1, is capable of forming amyloid fibrils in protein misfolding study, indicating a protective role of the SP. Altogether, our results demonstrated a novel role of the SP in SAA folding and misfolding and provided a novel direction for therapeutic development of AA amyloidosis.
Topics: Humans; Serum Amyloid A Protein; Protein Sorting Signals; Amyloidosis; Amyloid
PubMed: 36309973
DOI: 10.1002/pro.4485 -
ACS Synthetic Biology Feb 2023The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology...
The passage of proteins across biological membranes via the general secretory (Sec) pathway is a universally conserved process with critical functions in cell physiology and important industrial applications. Proteins are directed into the Sec pathway by a signal peptide at their N-terminus. Estimating the impact of physicochemical signal peptide features on protein secretion levels has not been achieved so far, partially due to the extreme sequence variability of signal peptides. To elucidate relevant features of the signal peptide sequence that influence secretion efficiency, an evaluation of ∼12,000 different designed signal peptides was performed using a novel miniaturized high-throughput assay. The results were used to train a machine learning model, and a post-hoc explanation of the model is provided. By describing each signal peptide with a selection of 156 physicochemical features, it is now possible to both quantify feature importance and predict the protein secretion levels directed by each signal peptide. Our analyses allow the detection and explanation of the relevant signal peptide features influencing the efficiency of protein secretion, generating a versatile tool for the de novo design and in silico evaluation of signal peptides.
Topics: Protein Sorting Signals; Bacillus subtilis; Protein Transport; Cell Membrane; Bacterial Proteins
PubMed: 36649479
DOI: 10.1021/acssynbio.2c00328 -
Molecular Microbiology May 2020The twin-arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates... (Review)
Review
The twin-arginine protein transport (Tat pathway) is found in prokaryotes and plant organelles and transports folded proteins across membranes. Targeting of substrates to the Tat system is mediated by the presence of an N-terminal signal sequence containing a highly conserved twin-arginine motif. The Tat machinery comprises membrane proteins from the TatA and TatC families. Assembly of the Tat translocon is dynamic and is triggered by the interaction of a Tat substrate with the Tat receptor complex. This review will summarise recent advances in our understanding of Tat transport, focusing in particular on the roles played by Tat signal peptides in protein targeting and translocation.
Topics: Amino Acid Motifs; Bacterial Proteins; Cell Membrane; Escherichia coli Proteins; Membrane Transport Proteins; Protein Binding; Protein Conformation; Protein Sorting Signals; Protein Transport; Twin-Arginine-Translocation System
PubMed: 31971282
DOI: 10.1111/mmi.14461