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Novel insights into brain glycogen metabolism.
Carlson GM, Dienel GA, Colbran RJ
(2018) J Biol Chem :
Show Abstract · Added March 21, 2018
The synthesis of glycogen allows for efficient intracellular storage of glucose molecules in a soluble form that can be rapidly released to enter glycolysis in response to energy demand. Intensive studies of glucose and glycogen metabolism, predominantly in skeletal muscle and liver, have produced innumerable insights into the mechanisms of hormone action, resulting in the award of several Nobel Prizes over the last one hundred years. Glycogen is actually present in all cells and tissues, albeit at much lower levels. However, metabolic and physiological roles of glycogen in other tissues are poorly understood. This series of mini-reviews summarizes what is known about the enzymes involved in brain glycogen metabolism and studies that have linked glycogen metabolism to multiple brain functions involving metabolic communication between astrocytes and neurons. Recent studies unexpectedly linking some forms of epilepsy to mutations in two poorly understood proteins involved in glycogen metabolism are also reviewed.
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
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Introduction to Metals in Biology 2018: Copper homeostasis and utilization in redox enzymes.
Guengerich FP
(2018) J Biol Chem :
Show Abstract · Added March 14, 2018
This 11th Thematic Metals in Biology Thematic Series deals with copper, a transition metal with a prominent role in biochemistry. Copper is a very versatile element, and both deficiencies and excesses can be problematic. Five Minireviews in this series deal with several aspects of copper homeostasis in microorganisms and mammals and the role of this metal in two enzymes, copper-only superoxide dismutase and cytochrome c oxidase.
Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
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Dual cyclooxygenase-fatty acid amide hydrolase inhibitor exploits novel binding interactions in the cyclooxygenase active site.
Goodman MC, Xu S, Rouzer CA, Banerjee S, Ghebreselasie K, Migliore M, Piomelli D, Marnett LJ
(2018) J Biol Chem 293: 3028-3038
Show Abstract · Added April 22, 2018
The cyclooxygenases COX-1 and COX-2 oxygenate arachidonic acid (AA) to prostaglandin H (PGH). COX-2 also oxygenates the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA) to the corresponding PGH analogs. Both enzymes are targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but NSAID-mediated COX inhibition is associated with gastrointestinal toxicity. One potential strategy to counter this toxicity is to also inhibit fatty acid amide hydrolase (FAAH), which hydrolyzes bioactive fatty acid ethanolamides (FAEs) into fatty acids and ethanolamine. Here, we investigated the mechanism of COX inhibition by ARN2508, an NSAID that inhibits both COXs and FAAH with high potency, target selectivity, and decreased gastrointestinal toxicity in mouse models, presumably due to its ability to increase levels of FAEs. A 2.27-Å-resolution X-ray crystal structure of the COX-2·()-ARN2508 complex reveals that ARN2508 adopts a binding pose similar to that of its parent NSAID flurbiprofen. However, ARN2508's alkyl tail is inserted deep into the top channel, an active site region not exploited by any previously reported NSAID. As for flurbiprofen, ARN2508's potency is highly dependent on the configuration of the α-methyl group. Thus, ()-ARN2508 is more potent than ()-ARN2508 for inhibition of AA oxygenation by both COXs and 2-AG oxygenation by COX-2. Also, similarly to ()-flurbiprofen, ()-ARN2508 exhibits substrate selectivity for inhibition of 2-AG oxygenation. Site-directed mutagenesis confirms the importance of insertion of the alkyl tail into the top channel for ()-ARN2508's potency and suggests a role for Ser-530 as a determinant of the inhibitor's slow rate of inhibition compared with that of ()-flurbiprofen.
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Inherent steroid 17α,20-lyase activity in defunct cytochrome P450 17A enzymes.
Gonzalez E, Johnson KM, Pallan PS, Phan TTN, Zhang W, Lei L, Wawrzak Z, Yoshimoto FK, Egli M, Guengerich FP
(2018) J Biol Chem 293: 541-556
Show Abstract · Added March 14, 2018
Cytochrome P450 (P450) 17A1 catalyzes the oxidations of progesterone and pregnenolone and is the major source of androgens. The enzyme catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction, and several mechanisms have been proposed for the latter step. Zebrafish P450 17A2 catalyzes only the 17α-hydroxylations. We previously reported high similarity of the crystal structures of zebrafish P450 17A1 and 17A2 and human P450 17A1. Five residues near the heme, which differed, were changed. We also crystallized this five-residue zebrafish P450 17A1 mutant, and the active site still resembled the structure in the other proteins, with some important differences. These P450 17A1 and 17A2 mutants had catalytic profiles more similar to each other than did the wildtype proteins. Docking with these structures can explain several minor products, which require multiple enzyme conformations. The 17α-hydroperoxy (OOH) derivatives of the steroids were used as oxygen surrogates. Human P450 17A1 and zebrafish P450s 17A1 and P450 17A2 readily converted these to the lyase products in the absence of other proteins or cofactors (with catalytically competent kinetics) plus hydroxylated 17α-hydroxysteroids. The 17α-OOH results indicate that a "Compound I" (FeO) intermediate is capable of formation and can be used to rationalize the products. We conclude that zebrafish P450 17A2 is capable of lyase activity with the 17α-OOH steroids because it can achieve an appropriate conformation for lyase catalysis in this system that is precluded in the conventional reaction.
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A neutralizing antibody that blocks delivery of the enzymatic cargo oftoxin TcdB into host cells.
Kroh HK, Chandrasekaran R, Zhang Z, Rosenthal K, Woods R, Jin X, Nyborg AC, Rainey GJ, Warrener P, Melnyk RA, Spiller BW, Lacy DB
(2018) J Biol Chem 293: 941-952
Show Abstract · Added March 15, 2018
infection is the leading cause of hospital-acquired diarrhea and is mediated by the actions of two toxins, TcdA and TcdB. The toxins perturb host cell function through a multistep process of receptor binding, endocytosis, low pH-induced pore formation, and the translocation and delivery of an N-terminal glucosyltransferase domain that inactivates host GTPases. Infection studies with isogenic strains having defined toxin deletions have established TcdB as an important target for therapeutic development. Monoclonal antibodies that neutralize TcdB function have been shown to protect againstinfection in animal models and reduce recurrence in humans. Here, we report the mechanism of TcdB neutralization by PA41, a humanized monoclonal antibody capable of neutralizing TcdB from a diverse array ofstrains. Through a combination of structural, biochemical, and cell functional studies, involving X-ray crystallography and EM, we show that PA41 recognizes a single, highly conserved epitope on the TcdB glucosyltransferase domain and blocks productive translocation and delivery of the enzymatic cargo into the host cell. Our study reveals a unique mechanism oftoxin neutralization by a monoclonal antibody, which involves targeting a process that is conserved across the large clostridial glucosylating toxins. The PA41 antibody described here provides a valuable tool for dissecting the mechanism of toxin pore formation and translocation across the endosomal membrane.
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Loss of αB-crystallin function in zebrafish reveals critical roles in the development of the lens and stress resistance of the heart.
Mishra S, Wu SY, Fuller AW, Wang Z, Rose KL, Schey KL, Mchaourab HS
(2018) J Biol Chem 293: 740-753
Show Abstract · Added April 3, 2018
Genetic mutations in the human small heat shock protein αB-crystallin have been implicated in autosomal cataracts and skeletal myopathies, including heart muscle diseases (cardiomyopathy). Although these mutations lead to modulation of their chaperone activity, thefunctions of αB-crystallin in the maintenance of both lens transparency and muscle integrity remain unclear. This lack of information has hindered a mechanistic understanding of these diseases. To better define the functional roles of αB-crystallin, we generated loss-of-function zebrafish mutant lines by utilizing the CRISPR/Cas9 system to specifically disrupt the two αB-crystallin genes, αand αWe observed lens abnormalities in the mutant lines of both genes, and the penetrance of the lens phenotype was higher in αthan αmutants. This finding is in contrast with the lack of a phenotype previously reported in αB-crystallin knock-out mice and suggests that the elevated chaperone activity of the two zebrafish orthologs is critical for lens development. Besides its key role in the lens, we uncovered another critical role for αB-crystallin in providing stress tolerance to the heart. The αB-crystallin mutants exhibited hypersusceptibility to develop pericardial edema when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our work illuminates the involvement of αB-crystallin in stress tolerance of the heart presumably through the proteostasis network and reinforces the critical role of the chaperone activity of αB-crystallin in the maintenance of lens transparency.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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Heterologous phosphorylation-induced formation of a stability lock permits regulation of inactive receptors by β-arrestins.
Tóth AD, Prokop S, Gyombolai P, Várnai P, Balla A, Gurevich VV, Hunyady L, Turu G
(2018) J Biol Chem 293: 876-892
Show Abstract · Added March 14, 2018
β-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, G-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded ATangiotensin receptor (ATR). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated ATR in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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Structure-function characterization of three human antibodies targeting the vaccinia virus adhesion molecule D8.
Matho MH, Schlossman A, Gilchuk IM, Miller G, Mikulski Z, Hupfer M, Wang J, Bitra A, Meng X, Xiang Y, Kaever T, Doukov T, Ley K, Crotty S, Peters B, Hsieh-Wilson LC, Crowe JE, Zajonc DM
(2018) J Biol Chem 293: 390-401
Show Abstract · Added March 14, 2018
Vaccinia virus (VACV) envelope protein D8 is one of three glycosaminoglycan adhesion molecules and binds to the linear polysaccharide chondroitin sulfate (CS). D8 is also a target for neutralizing antibody responses that are elicited by the smallpox vaccine, which has enabled the first eradication of a human viral pathogen and is a useful model for studying antibody responses. However, to date, VACV epitopes targeted by human antibodies have not been characterized at atomic resolution. Here, we characterized the binding properties of several human anti-D8 antibodies and determined the crystal structures of three VACV-mAb variants, VACV-66, VACV-138, and VACV-304, separately bound to D8. Although all these antibodies bound D8 with high affinity and were moderately neutralizing in the presence of complement, VACV-138 and VACV-304 also fully blocked D8 binding to CS-A, the low affinity ligand for D8. VACV-138 also abrogated D8 binding to the high-affinity ligand CS-E, but we observed residual CS-E binding was observed in the presence of VACV-304. Analysis of the VACV-138- and VACV-304-binding sites along the CS-binding crevice of D8, combined with different efficiencies of blocking D8 adhesion to CS-A and CS-E allowed us to propose that D8 has a high- and low-affinity CS-binding region within its central crevice. The crevice is amenable to protein engineering to further enhance both specificity and affinity of binding to CS-E. Finally, a wild-type D8 tetramer specifically bound to structures within the developing glomeruli of the kidney, which express CS-E. We propose that through structure-based protein engineering, an improved D8 tetramer could be used as a potential diagnostic tool to detect expression of CS-E, which is a possible biomarker for ovarian cancer.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
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The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.
Kolobova E, Roland JT, Lapierre LA, Williams JA, Mason TA, Goldenring JR
(2017) J Biol Chem 292: 20394-20409
MeSH Terms: A Kinase Anchor Proteins, Biomarkers, Cell Line, Centrosome, Cytoskeletal Proteins, Humans, Imaging, Three-Dimensional, Intracellular Signaling Peptides and Proteins, Luminescent Proteins, Microscopy, Electron, Transmission, Microtubule-Associated Proteins, Microtubule-Organizing Center, Models, Molecular, Nerve Tissue Proteins, Peptide Fragments, Phosphoproteins, Protein Interaction Domains and Motifs, Protein Interaction Mapping, Protein Multimerization, Proteomics, RNA Interference, Recombinant Fusion Proteins, Recombinant Proteins, Two-Hybrid System Techniques
Show Abstract · Added April 3, 2018
Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.
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Loss of hepatic AMP-activated protein kinase impedes the rate of glycogenolysis but not gluconeogenic fluxes in exercising mice.
Hughey CC, James FD, Bracy DP, Donahue EP, Young JD, Viollet B, Foretz M, Wasserman DH
(2017) J Biol Chem 292: 20125-20140
MeSH Terms: AMP-Activated Protein Kinases, Animals, Energy Metabolism, Gluconeogenesis, Glucose, Glycogenolysis, Homeostasis, Isotope Labeling, Liver, Mice, Mice, Knockout, Physical Conditioning, Animal
Show Abstract · Added March 14, 2018
Pathologies including diabetes and conditions such as exercise place an unusual demand on liver energy metabolism, and this demand induces a state of energy discharge. Hepatic AMP-activated protein kinase (AMPK) has been proposed to inhibit anabolic processes such as gluconeogenesis in response to cellular energy stress. However, both AMPK activation and glucose release from the liver are increased during exercise. Here, we sought to test the role of hepatic AMPK in the regulation ofglucose-producing and citric acid cycle-related fluxes during an acute bout of muscular work. We usedH/C metabolic flux analysis to quantify intermediary metabolism fluxes in both sedentary and treadmill-running mice. Additionally, liver-specific AMPK α1 and α2 subunit KO and WT mice were utilized. Exercise caused an increase in endogenous glucose production, glycogenolysis, and gluconeogenesis from phosphoenolpyruvate. Citric acid cycle fluxes, pyruvate cycling, anaplerosis, and cataplerosis were also elevated during this exercise. Sedentary nutrient fluxes in the postabsorptive state were comparable for the WT and KO mice. However, the increment in the endogenous rate of glucose appearance during exercise was blunted in the KO mice because of a diminished glycogenolytic flux. This lower rate of glycogenolysis was associated with lower hepatic glycogen content before the onset of exercise and prompted a reduction in arterial glucose during exercise. These results indicate that liver AMPKα1α2 is required for maintaining glucose homeostasis during an acute bout of exercise.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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12 MeSH Terms