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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 -
Applied Microbiology and Biotechnology Feb 2020In addition to the extracellular proteins secreted by known secretory pathways, a number of cytoplasmic proteins without predicable or known signal sequences or... (Review)
Review
In addition to the extracellular proteins secreted by known secretory pathways, a number of cytoplasmic proteins without predicable or known signal sequences or secretory motifs have been found in the extracellular milieu, and were consequently classified as non-classically secreted proteins. Non-classical protein secretion is considered to be a general, conserved cellular phenomenon in both eukaryotes and prokaryotes. There are several research hotspots on the non-classical protein secretory pathway, and the most important two of them are the recognition principle of substrate proteins and possible secretory mechanisms. To date, researchers have made some progress in understanding the characteristics of these proteins. For example, it was discovered that many non-classically secreted proteins exist and are secreted in multimeric form. Some of these proteins prefer to be clustered and exported at the poles and the septum of the cell. The majority of these proteins play different functions when they are in the intra- and extracellular environments, and several of their functions are related to survival and pathogenicity. Furthermore, non-classically secreted proteins can be used as leading proteins to guide a POI (protein of interest) out of the cells, which provides a novel strategy for protein secretion with potential applications in the industry. Summarizing these findings, this review emphasizes the hot spots related to non-classically secreted proteins in bacteria, lists the most important hypotheses on the selection and secretion mechanisms of non-classically secreted proteins, and put forward their potential applications.
Topics: Bacteria; Bacterial Proteins; Protein Transport; Secretory Pathway
PubMed: 31853566
DOI: 10.1007/s00253-019-10285-4 -
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 -
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 -
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 -
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 -
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 -
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 -
Optometry and Vision Science : Official... Jul 2015Within a few minutes of wear, contact lenses become rapidly coated with a variety of tear film components, including proteins, lipids, and mucins. Tears have a rich and... (Review)
Review
Within a few minutes of wear, contact lenses become rapidly coated with a variety of tear film components, including proteins, lipids, and mucins. Tears have a rich and complex composition, allowing a wide range of interactions and competitive processes, with the first event observed at the interface between a contact lens and tear fluid being protein adsorption. Protein adsorption on hydrogel contact lenses is a complex process involving a variety of factors relating to both the protein in question and the lens material. Among tear proteins, lysozyme is a major protein that has both antibacterial and anti-inflammatory functions. Contact lens materials that have high ionicity and high water content have an increased affinity to accumulate lysozyme during wear, when compared with other soft lens materials, notably silicone hydrogel lenses. This review provides an overview of tear film proteins, with a specific focus on lysozyme, and examines various factors that influence protein deposition on contact lenses. In addition, the impact of lysozyme deposition on various ocular physiological responses and bacterial adhesion to lenses and the interaction of lysozyme with other tear proteins are reviewed. This comprehensive review suggests that deposition of lysozyme on contact lens materials may provide a number of beneficial effects during contact lens wear.
Topics: Adsorption; Contact Lenses, Hydrophilic; Eye Proteins; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Muramidase; Protein Binding; Tears
PubMed: 26002002
DOI: 10.1097/OPX.0000000000000615 -
Biochimie Nov 2021Over the past decades, much have been learned about HIV-1 virus and its molecular strategies for pathogenesis. However, HIV-1 still remains an enigmatic virus,... (Review)
Review
Over the past decades, much have been learned about HIV-1 virus and its molecular strategies for pathogenesis. However, HIV-1 still remains an enigmatic virus, particularly because of its unique proteins. Establishment of latency and reactivation is still a puzzling question and various temporal and spatial dynamics between HIV-1 proteins itself have given us new way of thinking about its pathogenesis. HIV-1 replication depends on Tat which is a small unstructured protein and subjected to various post-translational modifications for its myriad of functions. HIV-1 Tat protein modulates the functions of various strategic cellular pathways like proteasomal machinery and inflammatory pathways to aid in HIV-1 pathogenesis. Many of the recent findings have shown that Tat is associated with exosomes, cleared from HIV-1 infected cells through its degradation by diverse routes ranging from lysosomal to proteasomal pathways. HIV-1 Tat was also found to be associated with other HIV-1 proteins including Vpr, Nef, Nucleocapsid (NC) and Rev. Interaction of Tat with Vpr and Nef increases its transactivation function, whereas, interaction of Tat with NC or Rev leads to Tat protein degradation and hence suppression of Tat functions. Research in the recent years has established that Tat is not only important for HIV-1 promoter transactivation and virus replication but also modulating multiple cellular and molecular functions leading to HIV-1 pathogenicity. In this review we discussed various transcriptional and non-transcriptional HIV-1 Tat functions which modulate host cell metabolism during HIV-1 pathogenesis.
Topics: Animals; Antigen Presentation; Apoptosis; Autophagy; Bodily Secretions; Gene Expression Regulation; HIV-1; Humans; Oxidative Stress; Proteolysis; tat Gene Products, Human Immunodeficiency Virus
PubMed: 34242726
DOI: 10.1016/j.biochi.2021.07.001