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The COPII coat complex, which mediates secretory cargo trafficking from the endoplasmic reticulum, is a key control point for subcellular protein targeting. Because misdirected proteins cannot function, protein sorting by COPII is critical for establishing and maintaining normal cell and tissue homeostasis. Indeed, mutations in COPII genes cause a range of human pathologies, including cranio-lenticulo-sutural dysplasia (CLSD), which is characterized by collagen trafficking defects, craniofacial abnormalities, and skeletal dysmorphology. Detailed knowledge of the COPII pathway is required to understand its role in normal cell physiology and to devise new treatments for disorders in which it is disrupted. However, little is known about how vertebrates dynamically regulate COPII activity in response to developmental, metabolic, or pathological cues. Several COPII proteins are modified by O-linked β-N-acetylglucosamine (O-GlcNAc), a dynamic form of intracellular protein glycosylation, but the biochemical and functional effects of these modifications remain unclear. Here, we use a combination of chemical, biochemical, cellular, and genetic approaches to demonstrate that site-specific O-GlcNAcylation of COPII proteins mediates their protein-protein interactions and modulates cargo secretion. In particular, we show that individual O-GlcNAcylation sites of SEC23A, an essential COPII component, are required for its function in human cells and vertebrate development, because mutation of these sites impairs SEC23A-dependent in vivo collagen trafficking and skeletogenesis in a zebrafish model of CLSD. Our results indicate that O-GlcNAc is a conserved and critical regulatory modification in the vertebrate COPII-dependent trafficking pathway.
AIMS - Ghrelin is a gastric-derived hormone that stimulates growth hormone (GH) secretion and has a multi-faceted role in the regulation of energy homeostasis, including glucose metabolism. Circulating ghrelin concentrations are modulated in response to nutritional status, but responses to ghrelin in altered metabolic states are poorly understood. We investigated the metabolic effects of ghrelin in obesity and early after Roux-en-Y gastric bypass (RYGB).
MATERIALS AND METHODS - We assessed central and peripheral metabolic responses to acyl ghrelin infusion (1 pmol kg min ) in healthy, lean subjects (n = 9) and non-diabetic, obese subjects (n = 9) before and 2 weeks after RYGB. Central responses were assessed by GH and pancreatic polypeptide (surrogate for vagal activity) secretion. Peripheral responses were assessed by hepatic and skeletal muscle insulin sensitivity during a hyperinsulinaemic-euglycaemic clamp.
RESULTS - Ghrelin-stimulated GH secretion was attenuated in obese subjects, but was restored by RYGB to a response similar to that of lean subjects. The heightened pancreatic polypeptide response to ghrelin infusion in the obese was attenuated after RYGB. Hepatic glucose production and hepatic insulin sensitivity were not altered by ghrelin infusion in RYGB subjects. Skeletal muscle insulin sensitivity was impaired to a similar degree in lean, obese and post-RYGB individuals in response to ghrelin infusion.
CONCLUSIONS - These data suggest that obesity is characterized by abnormal central, but not peripheral, responsiveness to ghrelin that can be restored early after RYGB before significant weight loss. Further work is necessary to fully elucidate the role of ghrelin in the metabolic changes that occur in obesity and following RYGB.
© 2017 John Wiley & Sons Ltd.
Lipid electrophiles modify cellular targets, altering their function. Here, we describe histones as major targets for modification by 4-oxo-2-nonenal, resulting in a stable Lys modification structurally analogous to other histone Lys acylations. Seven adducts were identified in chromatin isolated from intact cells: four 4-ketoamides to Lys and three Michael adducts to His. A 4-ketoamide adduct residing at H3K27 was identified in stimulated macrophages. Modification of histones H3 and H4 prevented nucleosome assembly.
Membrane proteins are abundant, critically important biomolecules that conduct essential functions in all cells and are the targets of a significant number of therapeutic drugs. However, the analysis of their expression, modification, protein-protein interactions, and structure by mass spectrometry has lagged behind similar studies of soluble proteins. Here we review the limitations to analysis of integral membrane and membrane-associated proteins and highlight advances in sample preparation and mass spectrometry methods that have led to the successful analysis of this protein class. Advances in the analysis of membrane protein posttranslational modification, protein-protein interaction, protein structure, and tissue distributions by imaging mass spectrometry are discussed. Furthermore, we focus our discussion on the application of mass spectrometry for the analysis of aquaporins as a prototypical integral membrane protein and how advances in analytical methods have revealed new biological insights into the structure and function of this family of proteins.
Fatty acid acylation of proteins is a well-studied co- or posttranslational modification typically conferring membrane trafficking signals or membrane anchoring properties to proteins. Commonly observed examples of protein acylation include N-terminal myristoylation and palmitoylation of cysteine residues. In the present study, direct tissue profiling mass spectrometry of bovine and human lens sections revealed an abundant signal tentatively assigned as a lipid-modified form of aquaporin-0. LC/MS/MS proteomic analysis of hydrophobic tryptic peptides from lens membrane proteins revealed both N-terminal and C-terminal peptides modified by 238 and 264 Da which were subsequently assigned by accurate mass measurement as palmitoylation and oleoylation, respectively. Specific sites of modification were the N-terminal methionine residue and lysine 238 revealing, for the first time, an oleic acid modification via an amide linkage to a lysine residue. The specific fatty acids involved reflect their abundance in the lens fiber cell plasma membrane. Imaging mass spectrometry indicated abundant acylated AQP0 in the inner cortical region of both bovine and human lenses and acylated truncation products in the lens nucleus. Additional analyses revealed that the lipid-modified forms partitioned exclusively to a detergent-resistant membrane fraction, suggesting a role in membrane domain targeting.
Comprehensive lipid profiling by mass spectrometry provides comparative data on the relative distribution of individual glycerophospholipids within each of the major classes. Application of this method to the analysis of glycerophospholipid remodeling in murine primary resident peritoneal macrophages (RPMs) during zymosan phagocytosis reveals significant decreases in the levels of every major arachidonic acid (20:4)-containing species of phosphatidylcholine (GPCho) and in selected 20:4-containing phosphatidylinositol (GPIns) and phosphatidylglycerol (GPGro) species. No net changes in 20:4-containing phosphatidylethanolamine (GPEtn) species were detected. Pretreatment of RPMs with LPS resulted in subtle changes in the magnitude and kinetics of the response but had no effect on the overall pattern of zymosan-induced glycerophospholipid remodeling. Inhibition of prostaglandin (PG) synthesis with indomethacin reduced the magnitude of the changes in 20:4-containing diacyl but not alkyl acyl species. Blockade of 20:4 reacylation with thimerosal had no effect on the magnitude of the zymosan-induced changes in GPCho, GPIns, or GPGro species but revealed decreases in the level of alkyl acyl GEtn species. RAW264.7 cells contain much lower levels of phospholipid 20:4 than do RPMs and synthesize PGs poorly in response to zymosan. Pretreatment with granulocyte-macrophage colony stimulating factor, lipopolysaccharide, and interferon-gamma substantially increased the extent of 20:4 mobilization and PG synthesis in these cells. However, under conditions of maximal zymosan-dependent PG synthesis, the only glycerophospholipid that exhibited a significant change was a 20:4-containing plasmenyl GPEtn. These results suggest that GPCho is the major ultimate source of 20:4 that is mobilized in zymosan-stimulated RPMs but that 20:4 mobilization may involve the intermediate turnover of alkyl acyl GPEtn species.
A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.
Bovine thrombin and human factor Xa were acylated at their active site selectively with inhibitors derived from the parent compound 4-guanidinophenyl (E)-4-diethylamino-2-hydroxy-alpha-methylcinnamate hydrochloride, 1b. Peptidyl side chains were attached to the phenol ring via amide connection, which served as a recognition motif in inhibiting different serine proteases. Upon irradiation with 366 nm light, the trans-cinnamate attached to the active-site serine isomerizes to the cis isomer which then rapidly lactonizes to release the free enzyme. The peptidyl side chain sequences specific for each serine protease were revealed via constructing and screening a library of homologous compounds. This methodology may be applied to other proteases. One application based on enzyme-specific, photoactivatable inhibitors is to isolate a designated active protease from a mixture of several proteases. Thus, a cinnamate inhibitor with a biotin moiety, 1d, was synthesized. A solution of enzyme-specific, biotinylated inhibitor was added into a mixture of proteases containing a target enzyme. The target enzyme was acylated at the active site and subsequently bore a biotin tail. An avidin column was used to separate the biotinylated enzyme from the unmodified ones, by a strong binding between biotin and avidin. After a brief irradiation on the avidin column, the retained enzymes were released from the biotin tag and eluted off the column. To demonstrate the idea, thrombin and factor Xa have been separated from each other by this strategy.
Mannan interacts with mannose/N-acetylglucosamine (GlcNAc) receptors on the surface of both Kupffer cells and endothelial cells in the liver, and induces glycogenolysis through production of peptide-leukotriene (LT) in the perfused rat liver. In the present study, we examined whether positively and negatively charged GlcNAc-containing polysaccharides stimulate glycogenolysis in perfused rat liver. Infusion of the former, 67% deacetylated chitin (DAC), induced biphasic increases in glucose production and a steep decrease in oxygen consumption by the liver. ONO-1078, an LT D4 receptor antagonist, abolished the suppression of oxygen consumption and reduced the glucose production by DAC. Infusion of the latter, hyaluronan, stimulated glucose production with a concomitant increase in oxygen consumption. Ibuprofen, a cyclooxygenase inhibitor, reduced the glucose production by hyaluronan. Sequential infusions of mannan and DAC, but not hyaluronan, did not induce glycogenolytic responses when mannan was infused 20 min before the second stimulation. These results suggest that DAC, but not hyaluronan, stimulates mannose/GlcNAc receptors in the perfused rat liver, and that potent immunological activity induced by DAC may be mediated by activation of the receptors.
Derivatives of the potent antiinflammatory agent and cyclooxygenase inhibitor indomethacin were synthesized in which the carboxylic acid moiety was converted into reactive acylating agents. Indomethacin imidazole (indomethacin-IM) and indomethacin N-hydroxysuccinimide (indomethacin-NHS) inactivated both the cyclooxygenase and peroxidase activities when incubated with the apo form of purified prostaglandin endoperoxide synthase (PGH synthase) at a stoichiometry of 1:1. Treatment of the inactivated enzyme with hydroxylamine at neutral pH led to recovery of all peroxidase and about 50% of the cyclooxygenase activity. Hydroxylamine did not regenerate the cyclooxygenase activity of indomethacin-inactivated protein. Reconstitution of the apoprotein with heme protected against inactivation by indomethacin-NHS. Visible spectroscopy established that indomethacin-NHS-inactivated apoenzyme had a reduced capacity to bind heme. Indomethacin-NHS also substantially protected the apoenzyme from cleavage at the trypsin-sensitive Arg277 site. Incubation of [2-14C]indomethacin-NHS with PGH synthase led to incorporation of radioactivity into the protein, but no adduct was detected by reversed-phase HPLC, suggesting it was unstable to the chromatographic conditions. Incubation of indomethacin-NHS with apoprotein followed by HPLC analysis led to the formation of greater amounts of the hydrolysis product indomethacin than did similar treatment of holoprotein. The results suggest that indomethacin-IM and indomethacin-NHS covalently and selectively label PGH synthase near the heme binding site, leading to loss of both catalytic activities of the enzyme.