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International Journal of Molecular... Jun 2022Many heterologous proteins can be secreted by bacterial ATP-binding cassette (ABC) transporters, provided that they are fused with the C-terminal signal sequence, but...
Many heterologous proteins can be secreted by bacterial ATP-binding cassette (ABC) transporters, provided that they are fused with the C-terminal signal sequence, but some proteins are not secretable even though they carry the right signal sequence. The invention of a method to secrete these non-secretable proteins would be valuable both for understanding the secretory physiology of ABC transporters and for industrial applications. Herein, we postulate that cationic "supercharged" regions within the target substrate protein block the secretion by ABC transporters. We also suggest that the secretion of such substrate proteins can be rescued by neutralizing those cationic supercharged regions via structure-preserving point mutageneses. Surface-protruding, non-structural cationic amino acids within the cationic supercharged regions were replaced by anionic or neutral hydrophilic amino acids, reducing the cationic charge density. The examples of rescued secretions we provide include the spike protein of SARS-CoV-2, glutathione-S-transferase, streptavidin, lipase, tyrosinase, cutinase, growth factors, etc. In summary, our study provides a method to predict the secretability and a tool to rescue the secretion by correcting the secretion-blocking regions, making a significant step in understanding the physiological properties of ABC transporter-dependent protein secretion and laying the foundation for the development of a secretion-based protein-producing platform.
Topics: ATP-Binding Cassette Transporters; Amino Acids; Bacterial Proteins; COVID-19; Humans; Protein Sorting Signals; SARS-CoV-2
PubMed: 35743142
DOI: 10.3390/ijms23126700 -
Nature Chemistry Sep 2022The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human...
The composition of soluble toxic protein aggregates formed in vivo is currently unknown in neurodegenerative diseases, due to their ultra-low concentration in human biofluids and their high degree of heterogeneity. Here we report a method to capture amyloid-containing aggregates in human biofluids in an unbiased way, a process we name amyloid precipitation. We use a structure-specific chemical dimer, a Y-shaped, bio-inspired small molecule with two capture groups, for amyloid precipitation to increase affinity. Our capture molecule for amyloid precipitation (CAP-1) consists of a derivative of Pittsburgh Compound B (dimer) to target the cross β-sheets of amyloids and a biotin moiety for surface immobilization. By coupling CAP-1 to magnetic beads, we demonstrate that we can target the amyloid structure of all protein aggregates present in human cerebrospinal fluid, isolate them for analysis and then characterize them using single-molecule fluorescence imaging and mass spectrometry. Amyloid precipitation enables unbiased determination of the molecular composition and structural features of the in vivo aggregates formed in neurodegenerative diseases.
Topics: Amyloid; Amyloid beta-Peptides; Bodily Secretions; Humans; Protein Aggregates
PubMed: 35798951
DOI: 10.1038/s41557-022-00976-3 -
PLoS Genetics Jul 2019The localization of mRNAs encoding secreted/membrane proteins (mSMPs) to the endoplasmic reticulum (ER) likely facilitates the co-translational translocation of secreted...
The localization of mRNAs encoding secreted/membrane proteins (mSMPs) to the endoplasmic reticulum (ER) likely facilitates the co-translational translocation of secreted proteins. However, studies have shown that mSMP recruitment to the ER in eukaryotes can occur in a manner that is independent of the ribosome, translational control, and the signal recognition particle, although the mechanism remains largely unknown. Here, we identify a cis-acting RNA sequence motif that enhances mSMP localization to the ER and appears to increase mRNA stability, and both the synthesis and secretion of secretome proteins. Termed SECReTE, for secretion-enhancing cis regulatory targeting element, this motif is enriched in mRNAs encoding secretome proteins translated on the ER in eukaryotes and on the inner membrane of prokaryotes. SECReTE consists of ≥10 nucleotide triplet repeats enriched with pyrimidine (C/U) every third base (i.e. NNY, where N = any nucleotide, Y = pyrimidine) and can be present in the untranslated as well as the coding regions of the mRNA. Synonymous mutations that elevate the SECReTE count in a given mRNA (e.g. SUC2, HSP150, and CCW12) lead to an increase in protein secretion in yeast, while a reduction in count led to less secretion and physiological defects. Moreover, the addition of SECReTE to the 3'UTR of an mRNA for an exogenously expressed protein (e.g. GFP) led to its increased secretion from yeast cells. Thus, SECReTE constitutes a novel RNA motif that facilitates ER-localized mRNA translation and protein secretion.
Topics: 3' Untranslated Regions; Endoplasmic Reticulum; Fungal Proteins; Nucleotide Motifs; Protein Biosynthesis; RNA Stability; RNA Transport; RNA, Fungal; RNA, Messenger; Saccharomyces cerevisiae; Silent Mutation
PubMed: 31260446
DOI: 10.1371/journal.pgen.1008248 -
Translational Vision Science &... Dec 2022To screen and compare the differential proteins in meibomian gland secretions between patients with blepharokeratoconjunctivitis (BKC) and healthy individuals and to...
PURPOSE
To screen and compare the differential proteins in meibomian gland secretions between patients with blepharokeratoconjunctivitis (BKC) and healthy individuals and to identify target proteins that may participate in the occurrence and development of BKC.
METHODS
Thirteen patients diagnosed with BKC in Shenzhen Eye Hospital and five healthy volunteers were included in this study. Meibomian gland secretions and clinical traits were collected before and after 1 month of standard BKC treatment. Label-free mass spectrometry was used for proteomic detection of meibomian gland secretions. Weighted protein coexpression network analysis (WPCNA) and several different protein analyses were performed to identify hub proteins associated with BKC and its clinical characteristics.
RESULTS
Patients with BKC had significantly lower cleanliness of the eyelid margin, higher palpebral margin scores, more serious clinical manifestations of secretions, and more damaged meibomian gland morphology compared with the healthy controls. One hundred fifteen differential proteins were associated with the clinical traits, which included diagnosis, sex, age, severity, corneal neovascularization, disease course, eyelid margin cleanliness, palpebral margin score, secretion characteristics, and meibomian gland morphology. Four hub proteins related to inflammation and the immune response (namely, S100A8, S100A9, ANXA3, and LCN2) were increased in BKC and remained increased after 1 month of treatment. The cleanliness, blepharon eyelid score, and secretion characteristics were improved after BKC treatment.
CONCLUSIONS
S100A8, S100A9, ANXA3, and LCN2 are BKC-associated proteins probably involved in the chronic inflammation of BKC.
TRANSLATIONAL RELEVANCE
Hub proteins probably involved in chronic inflammation of BKC were identified by proteomic methods.
Topics: Humans; Meibomian Glands; Proteomics; Bodily Secretions; Calgranulin A; Calgranulin B; Inflammation
PubMed: 36458945
DOI: 10.1167/tvst.11.12.4 -
Journal of Hepatology Feb 2021Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical... (Review)
Review
Many epithelia secrete bicarbonate-rich fluid to generate flow, alter viscosity, control pH and potentially protect luminal and intracellular structures from chemical stress. Bicarbonate is a key component of human bile and impaired biliary bicarbonate secretion is associated with liver damage. Major efforts have been undertaken to gain insight into acid-base homeostasis in cholangiocytes and more can be learned from analogous secretory epithelia. Extrahepatic examples include salivary and pancreatic duct cells, duodenocytes, airway and renal epithelial cells. The cellular machinery involved in acid-base homeostasis includes carbonic anhydrase enzymes, transporters of the solute carrier family, and intra- and extracellular pH sensors. This pH-regulatory system is orchestrated by protein-protein interactions, the establishment of an electrochemical gradient across the plasma membrane and bicarbonate sensing of the intra- and extracellular compartment. In this review, we discuss conserved principles identified in analogous secretory epithelia in the light of current knowledge on cholangiocyte physiology. We present a framework for cholangiocellular acid-base homeostasis supported by expression analysis of publicly available single-cell RNA sequencing datasets from human cholangiocytes, which provide insights into the molecular basis of pH homeostasis and dysregulation in the biliary system.
Topics: Acid-Base Equilibrium; Acid-Base Imbalance; Bicarbonates; Bile; Bile Ducts; Epithelium; Humans; Hydrogen-Ion Concentration; Secretory Pathway
PubMed: 33342564
DOI: 10.1016/j.jhep.2020.10.010 -
Neuroendocrinology 2023Identification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to... (Review)
Review
Identification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative, or metabolic diseases. To visualize molecule distribution and dynamics at the nanoscale, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies using transmission electron microscopy, atomic force microscopy, total internal reflection microscopy, and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein or protein-lipid interactions. Furthermore, the potential and the challenges of these different advanced imaging approaches for application in the study of neuroendocrine cell biology are discussed, aiming to guide researchers to select the best approach for their specific purpose around the crucial but not yet fully understood neurosecretion process.
Topics: Exocytosis; Bodily Secretions; Diagnostic Imaging
PubMed: 34915491
DOI: 10.1159/000521457 -
Experimental Physiology Apr 2010Intestinal fluid secretion is pivotal in the creation of an ideal environment for effective enzymatic digestion, nutrient absorption and stool movement. Since fluid... (Review)
Review
Intestinal fluid secretion is pivotal in the creation of an ideal environment for effective enzymatic digestion, nutrient absorption and stool movement. Since fluid cannot be actively secreted into the gut, this process is dependent on an osmotic gradient, which is mainly created by chloride transport by the enterocyte. A pathological dysbalance between fluid secretion and absorption leads to obstruction or potentially fatal diarrhoea. This article reviews the widely accepted model of intestinal chloride secretion with an emphasis on the molecular players involved in this tightly regulated process.
Topics: Animals; CLC-2 Chloride Channels; Chloride Channels; Chlorides; Cystic Fibrosis Transmembrane Conductance Regulator; Diarrhea; Enterocytes; Humans; Intestinal Absorption; Intestinal Mucosa; Intestinal Secretions; Potassium Channels; Sodium-Potassium-Chloride Symporters; Sodium-Potassium-Exchanging ATPase; Tight Junctions; Water-Electrolyte Balance
PubMed: 20233891
DOI: 10.1113/expphysiol.2009.049445 -
Journal of Extracellular Vesicles Sep 2022Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling...
Extracellular vesicle (EV) secretion enables cell-cell communication in multicellular organisms. During development, EV secretion and the specific loading of signalling factors in EVs contributes to organ development and tissue differentiation. Here, we present an in vivo model to study EV secretion using the fat body and the haemolymph of the fruit fly, Drosophila melanogaster. The system makes use of tissue-specific EV labelling and is amenable to genetic modification by RNAi. This allows the unique combination of microscopic visualisation of EVs in different organs and quantitative biochemical purification to study how EVs are generated within the cells and which factors regulate their secretion in vivo. Characterisation of the system revealed that secretion of EVs from the fat body is mainly regulated by Rab11 and Rab35, highlighting the importance of recycling Rab GTPase family members for EV secretion. We furthermore discovered a so far unknown function of Rab14 along with the kinesin Klp98A in EV biogenesis and secretion.
Topics: Animals; Bodily Secretions; Drosophila Proteins; Drosophila melanogaster; Endosomes; Extracellular Vesicles; Kinesins; Signal Transduction; rab GTP-Binding Proteins
PubMed: 36103151
DOI: 10.1002/jev2.12263 -
Cell Reports Jun 2020Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly...
Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in β cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.
Topics: Animals; Biological Transport; Bodily Secretions; Cell Communication; Glucagon; Glucagon-Secreting Cells; Humans; Mice, Knockout; Phenotype; Vitamin D; Vitamin D-Binding Protein
PubMed: 32553153
DOI: 10.1016/j.celrep.2020.107761 -
Bone Research Aug 2023Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of...
Proper regulation of Wnt signaling is critical for normal bone development and homeostasis. Mutations in several Wnt signaling components, which increase the activity of the pathway in the skeleton, cause high bone mass in human subjects and mouse models. Increased bone mass is often accompanied by severe headaches from increased intracranial pressure, which can lead to fatality and loss of vision or hearing due to the entrapment of cranial nerves. In addition, progressive forehead bossing and mandibular overgrowth occur in almost all subjects. Treatments that would provide symptomatic relief in these subjects are limited. Porcupine-mediated palmitoylation is necessary for Wnt secretion and binding to the frizzled receptor. Chemical inhibition of porcupine is a highly selective method of Wnt signaling inhibition. We treated three different mouse models of high bone mass caused by aberrant Wnt signaling, including homozygosity for loss-of-function in Sost, which models sclerosteosis, and two strains of mice carrying different point mutations in Lrp5 (equivalent to human G171V and A214V), at 3 months of age with porcupine inhibitors for 5-6 weeks. Treatment significantly reduced both trabecular and cortical bone mass in all three models. This demonstrates that porcupine inhibition is potentially therapeutic for symptomatic relief in subjects who suffer from these disorders and further establishes that the continued production of Wnts is necessary for sustaining high bone mass in these models.
Topics: Animals; Humans; Mice; Adaptor Proteins, Signal Transducing; Bodily Secretions; Disease Models, Animal; Gain of Function Mutation; Hyperostosis; Low Density Lipoprotein Receptor-Related Protein-5; Mutation
PubMed: 37612291
DOI: 10.1038/s41413-023-00278-5