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Biochemistry Jul 2020Myonectin/erythroferrone (also known as CTRP15) is a secreted hormone with metabolic function and a role in stress erythropoiesis. Despite its importance in physiologic...
Myonectin/erythroferrone (also known as CTRP15) is a secreted hormone with metabolic function and a role in stress erythropoiesis. Despite its importance in physiologic processes, biochemical characterization of the protein is lacking. Here, we show that multiple protein modifications are critical for myonectin secretion and multimerization. Abolishing N-linked glycosylation by tunicamycin, glucosamine supplementation, or glutamine substitutions of all four potential Asn glycosylation sites blocked myonectin secretion. Mass spectrometry confirmed that Asn-229 and Asn-281 were glycosylated, and substituting both Asn sites with Gln prevented myonectin secretion. Although Asn-319 is not identified as glycosylated, Gln substitution caused protein misfolding and retention in the endoplasmic reticulum. Of the four conserved cysteines, Cys-273 and Cys-278 were required for proper protein folding; Ala substitution of either site inhibited protein secretion. In contrast, Ala substitutions of Cys-142, Cys-194, or both markedly enhanced protein secretion, suggesting endoplasmic reticulum retention that facilitates myonectin oligomer assembly. Secreted myonectin consists of trimers, hexamers, and high-molecular weight (HMW) oligomers. The formation of higher-order structures via intermolecular disulfide bonds depended on Cys-142 and Cys-194; while the C142A mutant formed almost exclusively trimers, the C194A mutant was impaired in HMW oligomer formation. Most Pro residues within the short collagen domain of myonectin were also hydroxylated, a modification that stabilized the collagen triple helix. Inhibiting Pro hydroxylation or deleting the collagen domain markedly reduced the rate of protein secretion. Together, our results reveal key determinants that are important for myonectin folding, secretion, and multimeric assembly and provide a basis for future structure-function studies.
Topics: Animals; Cytokines; Glycosylation; HEK293 Cells; Humans; Hydroxylation; Mice; Muscle Proteins; Protein Folding; Protein Multimerization
PubMed: 32602701
DOI: 10.1021/acs.biochem.0c00461 -
Current Opinion in Structural Biology Apr 2023Contrary to first appearances, mucus structural biology is not an oxymoron. Though mucus hydrogels derive their characteristics largely from intrinsically disordered,... (Review)
Review
Contrary to first appearances, mucus structural biology is not an oxymoron. Though mucus hydrogels derive their characteristics largely from intrinsically disordered, heavily glycosylated polypeptide segments, the secreted mucin glycoproteins that constitute mucus undergo an orderly assembly process controlled by folded domains at their termini. Recent structural studies revealed how mucin complexes promote disulphide-mediated polymerization to produce the mucus gel scaffold. Additional protein-protein and protein-glycan interactions likely tune the mesoscale properties, stability, and activities of mucins. Evidence is emerging that even intrinsically disordered glycosylated segments have specific structural roles in the production and properties of mucus. Though soft-matter biophysical approaches to understanding mucus remain highly relevant, high-resolution structural studies of mucins and other mucus components are providing new perspectives on these vital, protective hydrogels.
Topics: Mucins; Mucus; Glycoproteins; Polysaccharides; Glycosylation
PubMed: 36753925
DOI: 10.1016/j.sbi.2022.102524 -
The Journal of Cell Biology May 2023Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined...
Secreted proteins fulfill a vast array of functions, including immunity, signaling, and extracellular matrix remodeling. In the trans-Golgi network, proteins destined for constitutive secretion are sorted into post-Golgi carriers which fuse with the plasma membrane. The molecular machinery involved is poorly understood. Here, we have used kinetic trafficking assays and transient CRISPR-KO to study biosynthetic sorting from the Golgi to the plasma membrane. Depletion of all canonical exocyst subunits causes cargo accumulation in post-Golgi carriers. Exocyst subunits are recruited to and co-localize with carriers. Exocyst abrogation followed by kinetic trafficking assays of soluble cargoes results in intracellular cargo accumulation. Unbiased secretomics reveals impairment of soluble protein secretion after exocyst subunit knockout. Importantly, in specialized cell types, the loss of exocyst prevents constitutive secretion of antibodies in lymphocytes and of leptin in adipocytes. These data identify exocyst as the functional tether of secretory post-Golgi carriers at the plasma membrane and an essential component of the mammalian constitutive secretory pathway.
Topics: Animals; Secretory Pathway; Exocytosis; Protein Transport; Golgi Apparatus; trans-Golgi Network; Proteins; Cell Membrane; Mammals
PubMed: 36920342
DOI: 10.1083/jcb.202205137 -
Nutrients Mar 2021The gastrointestinal tract can assess the nutrient composition of ingested food. The nutrient-sensing mechanisms in specialised epithelial cells lining the... (Review)
Review
The gastrointestinal tract can assess the nutrient composition of ingested food. The nutrient-sensing mechanisms in specialised epithelial cells lining the gastrointestinal tract, the enteroendocrine cells, trigger the release of gut hormones that provide important local and central feedback signals to regulate nutrient utilisation and feeding behaviour. The evidence for nutrient-stimulated secretion of two of the most studied gut hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), along with the known cellular mechanisms in enteroendocrine cells recruited by nutrients, will be the focus of this review. The mechanisms involved range from electrogenic transporters, ion channel modulation and nutrient-activated G-protein coupled receptors that converge on the release machinery controlling hormone secretion. Elucidation of these mechanisms will provide much needed insight into postprandial physiology and identify tractable dietary approaches to potentially manage nutrition and satiety by altering the secreted gut hormone profile.
Topics: Bodily Secretions; Enteroendocrine Cells; Gastric Inhibitory Polypeptide; Gastrointestinal Hormones; Gastrointestinal Tract; Glucagon-Like Peptide 1; Humans; Nutritional Physiological Phenomena; Postprandial Period
PubMed: 33803183
DOI: 10.3390/nu13030883 -
Sichuan Da Xue Xue Bao. Yi Xue Ban =... May 2023Saliva, a complex mixed biological fluid secreted by the salivary glands in the oral cavity, contains a wide variety of substances and information. With the development... (Review)
Review
Saliva, a complex mixed biological fluid secreted by the salivary glands in the oral cavity, contains a wide variety of substances and information. With the development of saliva omics, studies have shown that saliva not only serves as a huge reservoir of biomarker, but saliva diagnostics has also become a new diagnostic technology with the advantages of non-invasiveness, easy access, and low cost. However, finding "true" saliva biomarkers is still a challenge due to the complex and changeable nature of the oral environment and the high susceptibility of biomarker content to influences. Herein, mainly focusing on potential salivary biomarkers of common tumors, including DNA, RNA, proteins, metabolites and microorganisms, we gave a systematic overview of the biomarkers that had been identified so far or the associated biomarkers. We suggested that the future development direction should be the establishment of a multidisciplinary system for developing saliva diagnosis technology, the gradual construction of a saliva diagnosis platform, and the search for more precise pre-warning tumor biomarkers.
Topics: Humans; Biomarkers, Tumor; Biomarkers; Neoplasms; Saliva; Proteins
PubMed: 37248569
DOI: 10.12182/20230560112 -
Journal of Proteome Research Jan 2021Protein -acylation (commonly known as palmitoylation) is a widespread reversible lipid modification, which plays critical roles in regulating protein localization,... (Review)
Review
Protein -acylation (commonly known as palmitoylation) is a widespread reversible lipid modification, which plays critical roles in regulating protein localization, activity, stability, and complex formation. The deregulation of protein -acylation contributes to many diseases such as cancer and neurodegenerative disorders. The past decade has witnessed substantial progress in proteomic analysis of protein -acylation, which significantly advanced our understanding of -acylation biology. In this review, we summarized the techniques for the enrichment of -acylated proteins or peptides, critically reviewed proteomic studies of protein -acylation at eight different levels, and proposed major challenges for the -acylproteomics field. In summary, proteome-scale analysis of protein -acylation comes of age and will play increasingly important roles in discovering new disease mechanisms, biomarkers, and therapeutic targets.
Topics: Acylation; Lipoylation; Protein S; Proteome; Proteomics
PubMed: 33253586
DOI: 10.1021/acs.jproteome.0c00409 -
Molecules (Basel, Switzerland) Jun 2021Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes... (Review)
Review
Proteomics is a new area of study that in recent decades has provided great advances in the field of medicine. However, its enormous potential for the study of proteomes makes it also applicable to other areas of science. Milk is a highly heterogeneous and complex fluid, where there are numerous genetic variants and isoforms with post-translational modifications (PTMs). Due to the vast number of proteins and peptides existing in its matrix, proteomics is presented as a powerful tool for the characterization of milk samples and their products. The technology developed to date for the separation and characterization of the milk proteome, such as two-dimensional gel electrophoresis (2DE) technology and especially mass spectrometry (MS) have allowed an exhaustive characterization of the proteins and peptides present in milk and dairy products with enormous applications in the industry for the control of fundamental parameters, such as microbiological safety, the guarantee of authenticity, or the control of the transformations carried out, aimed to increase the quality of the final product.
Topics: Animals; Dairy Products; Electrophoresis, Gel, Two-Dimensional; Female; Mass Spectrometry; Mastitis; Milk; Milk Proteins; Peptides; Protein Processing, Post-Translational; Proteome; Proteomics; Quality Control
PubMed: 34201770
DOI: 10.3390/molecules26133832 -
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 -
Animal Science Journal = Nihon Chikusan... 2021The first secretion, 24-h post parturition of the mammary glands of sows, known as colostrum, is high in protein and low in lactose and fat. As a consequence of an... (Review)
Review
The first secretion, 24-h post parturition of the mammary glands of sows, known as colostrum, is high in protein and low in lactose and fat. As a consequence of an insufficient ingestion of colostrum, more than 50% of piglets fail to reach weaning and die. The composition and some functions of colostrum have been previously reported. For example, colostrum carbohydrates consist of mainly lactose. Lipids in the colostrum are mostly triacylglycerols, but <1% is fatty acids, which may act as homeostasis regulators. Similarly, proteins are found mostly as casein and whey, the latter being ≥80% immunoglobulins. Colostrum-derived immunoglobulins and bioactive proteins such as azurocidin help the immune system of the piglet fend off infections. In addition, leukocytes and exosomes are other minor but nonetheless equally crucial bioactive components in the porcine colostrum. Modern pig farming has achieved increases in pig productivity and litter size, but this has been accomplished in detriment of the health and the survival rate of piglets. Therefore, porcine colostrum is now even more important in pig farming. In the present review, we discuss the current knowledge on the composition and physiological functions of the porcine colostrum and briefly propose future research directions.
Topics: Animal Nutritional Physiological Phenomena; Animals; Animals, Newborn; Antimicrobial Cationic Peptides; Blood Proteins; Caseins; Colostrum; Diet; Exosomes; Female; Humans; Immunoglobulins; Infant; Lactose; Leukocytes; Litter Size; Parturition; Swine; Triglycerides; Weaning; Whey
PubMed: 34409709
DOI: 10.1111/asj.13618 -
Current Opinion in Structural Biology Aug 2021The bacterial outer membrane forms an impermeable barrier to the environment, but a wide variety of substances must cross it without compromising the membrane. Perhaps,... (Review)
Review
The bacterial outer membrane forms an impermeable barrier to the environment, but a wide variety of substances must cross it without compromising the membrane. Perhaps, the most fascinating transport phenomenon is the import and export of very large protein toxins using relatively small β-barrel proteins residing in the outer membrane. Progress has been made on three systems in recent years that shed light on this process. In this review, we summarize bacteriocin (toxin) import using TonB-dependent transporters and protein secretion by autotransporters and two partner secretion systems.
Topics: Bacterial Outer Membrane; Bacterial Outer Membrane Proteins; Bacterial Proteins; Biological Transport; Protein Transport
PubMed: 33901701
DOI: 10.1016/j.sbi.2021.03.007