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The Journal of Biological Chemistry Nov 2017Prion transmission between species is governed in part by primary sequence similarity between the infectious prion aggregate, PrP, and the cellular prion protein of the...
Prion transmission between species is governed in part by primary sequence similarity between the infectious prion aggregate, PrP, and the cellular prion protein of the host, PrP A puzzling feature of prion formation is that certain PrP sequences, such as that of bank vole, can be converted by a remarkably broad array of different mammalian prions, whereas others, such as rabbit, show robust resistance to cross-species prion conversion. To examine the structural determinants that confer susceptibility or resistance to prion conversion, we systematically tested over 40 PrP variants of susceptible and resistant PrP sequences in a prion conversion assay. Five key residue positions markedly impacted prion conversion, four of which were in steric zipper segments where side chains from amino acids tightly interdigitate in a dry interface. Strikingly, all five residue substitutions modulating prion conversion involved the gain or loss of an asparagine or glutamine residue. For two of the four positions, Asn and Gln residues were not interchangeable, revealing a strict requirement for either an Asn or Gln residue. Bank voles have a high number of Asn and Gln residues and a high Asn:Gln ratio. These findings suggest that a high number of Asn and Gln residues at specific positions may stabilize β-sheets and lower the energy barrier for cross-species prion transmission, potentially because of hydrogen bond networks from side chain amides forming extended Asn/Gln ladders. These data also suggest that multiple PrP segments containing Asn/Gln residues may act in concert along a replicative interface to promote prion conversion.
Topics: Amino Acid Substitution; Amyloid; Animals; Arvicolinae; Asparagine; Glutamine; Humans; Mice, Inbred C57BL; Models, Molecular; PrPC Proteins; Prion Diseases; Protein Conformation, beta-Strand; Protein Stability; Rabbits
PubMed: 28931606
DOI: 10.1074/jbc.M117.794107 -
Glycobiology Apr 2019Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that... (Review)
Review
Asparagine-linked (N-linked) glycosylation is one of the most common protein modification reactions in eukaryotic cells, occurring upon the majority of proteins that enter the secretory pathway. X-ray crystal structures of the single subunit OSTs from eubacterial and archaebacterial organisms revealed the location of donor and acceptor substrate binding sites and provided the basis for a catalytic mechanism. Cryoelectron microscopy structures of the octameric yeast OST provided substantial insight into the organization and assembly of the multisubunit oligosaccharyltransferases. Furthermore, the cryoelectron microscopy structure of a complex consisting of a mammalian OST complex, the protein translocation channel and a translating ribosome revealed new insight into the mechanism of cotranslational glycosylation.
Topics: Asparagine; Cryoelectron Microscopy; Crystallography, X-Ray; Eukaryotic Cells; Glycosylation; Hexosyltransferases; Humans; Membrane Proteins; Models, Molecular; Prokaryotic Cells; Protein Conformation
PubMed: 30312397
DOI: 10.1093/glycob/cwy093 -
MAbs 2022Deamidation of asparagine (Asn) and isomerization of aspartic acid (Asp) residues are among the most commonly observed spontaneous post-translational modifications...
Deamidation of asparagine (Asn) and isomerization of aspartic acid (Asp) residues are among the most commonly observed spontaneous post-translational modifications (PTMs) in proteins. Understanding and predicting a protein sequence's propensity for such PTMs can help expedite protein therapeutic discovery and development. In this study, we used proton-affinity calculations with semi-empirical quantum mechanics and microsecond long equilibrium molecular dynamics simulations to investigate mechanistic roles of structural conformation and chemical environment in dictating spontaneous degradation of Asn and Asp residues in 131 clinical-stage therapeutic antibodies. Backbone secondary structure, side-chain rotamer conformation and solvent accessibility were found to be key molecular indicators of Asp isomerization and Asn deamidation. Comparative analysis of backbone dihedral angles along with N-H proton affinity calculations provides a mechanistic explanation for the strong influence of the identity of the n + 1 residue on the rate of Asn/Asp degradation. With these findings, we propose a minimalistic physics-based classification model that can be leveraged to predict deamidation and isomerization propensity of proteins.
Topics: Isomerism; Protons; Asparagine; Aspartic Acid; Protein Structure, Secondary
PubMed: 36377085
DOI: 10.1080/19420862.2022.2143006 -
The Journal of Physical Chemistry. B Oct 2014Asparagine-linked carbohydrates profoundly impact glycoprotein folding, stability, and structure. However, the "glycosylation code" that relates these effects to protein...
Asparagine-linked carbohydrates profoundly impact glycoprotein folding, stability, and structure. However, the "glycosylation code" that relates these effects to protein sequence remains unsolved. We report atomically detailed replica exchange molecular dynamics simulations in explicit solvent that systematically investigate the impact of glycosylation upon peptides with the central sequon Pro-Asn-Gly/Ala-Thr-Trp/Ala. These simulations suggest that the effects of glycosylation may be quite sensitive to steric crowding by the side chain immediately following the glycosylation site but less sensitive to stacking interactions with the aromatic Trp residue. In addition, we compare our simulated ensembles with the known structures for full length glycoproteins. These structures corroborate the simulations and also suggest a remarkable consistency between the intraprotein and protein-glycan interactions of natural glycoproteins. Moreover, our analysis highlights the significance of left-handed conformations for compact β-hairpins at glycosylation sites. In summary, these studies elucidate basic biophysical principles for the glycosylation code.
Topics: Amino Acid Sequence; Asparagine; Computational Biology; Databases, Protein; Glycoproteins; Glycosylation; Molecular Dynamics Simulation; Peptides; Protein Structure, Secondary
PubMed: 25188817
DOI: 10.1021/jp508535f -
MBio Oct 2019As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose....
As obligate intracellular pathogens, viruses rely on the host cell machinery to replicate efficiently, with the host metabolism extensively manipulated for this purpose. High-throughput small interfering RNA (siRNA) screens provide a systematic approach for the identification of novel host-virus interactions. Here, we report a large-scale screen for host factors important for human cytomegalovirus (HCMV), consisting of 6,881 siRNAs. We identified 47 proviral factors and 68 antiviral factors involved in a wide range of cellular processes, including the mediator complex, proteasome function, and mRNA splicing. Focused characterization of one of the hits, asparagine synthetase (ASNS), demonstrated a strict requirement for asparagine for HCMV replication which leads to an early block in virus replication before the onset of DNA amplification. This effect is specific to HCMV, as knockdown of ASNS had little effect on herpes simplex virus 1 or influenza A virus replication, suggesting that the restriction is not simply due to a failure in protein production. Remarkably, virus replication could be completely rescued 7 days postinfection with the addition of exogenous asparagine, indicating that while virus replication is restricted at an early stage, it maintains the capacity for full replication days after initial infection. This study represents the most comprehensive siRNA screen for the identification of host factors involved in HCMV replication and identifies the nonessential amino acid asparagine as a critical factor in regulating HCMV virus replication. These results have implications for control of viral latency and the clinical treatment of HCMV in patients. HCMV accounts for more than 60% of complications associated with solid organ transplant patients. Prophylactic or preventative treatment with antivirals, such as ganciclovir, reduces the occurrence of early onset HCMV disease. However, late onset disease remains a significant problem, and prolonged treatment, especially in patients with suppressed immune systems, greatly increases the risk of antiviral resistance. Very few antivirals have been developed for use against HCMV since the licensing of ganciclovir, and of these, the same viral genes are often targeted, reducing the usefulness of these drugs against resistant strains. An alternative approach is to target host genes essential for virus replication. Here we demonstrate that HCMV replication is highly dependent on levels of the amino acid asparagine and that knockdown of a critical enzyme involved in asparagine synthesis results in severe attenuation of virus replication. These results suggest that reducing asparagine levels through dietary restriction or chemotherapeutic treatment could limit HCMV replication in patients.
Topics: Asparagine; Aspartate-Ammonia Ligase; Cells, Cultured; Cytomegalovirus; Fibroblasts; Gene Knockdown Techniques; Genetic Testing; Herpesvirus 1, Human; Host-Pathogen Interactions; Humans; Influenza A virus; Virus Replication
PubMed: 31594813
DOI: 10.1128/mBio.01651-19 -
MBio Aug 2017While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and...
While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway. We have previously found that Kaposi's sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.
Topics: Asparagine; Aspartate Aminotransferases; Aspartic Acid; Cell Proliferation; Glutamate Dehydrogenase; Glutamic Acid; Glutaminase; Glutamine; Herpesvirus 8, Human; Humans; Metabolic Networks and Pathways; Neoplasms; Nitrogen; Nucleotides
PubMed: 28811348
DOI: 10.1128/mBio.01179-17 -
Cell Chemical Biology May 2016Every month the editors of Cell Chemical Biology bring you highlights of the most recent chemical biology literature. Our May 2016 selection includes a new method for...
Every month the editors of Cell Chemical Biology bring you highlights of the most recent chemical biology literature. Our May 2016 selection includes a new method for labeling and visualizing nonprotein biomolecules using electron microscopy; a small molecule, originally found in tangerine peel, that regulates our circadian clock and helps with some of the metabolic pathologies associated with circadian rhythms; and a finding that asparagine is used as an amino acid exchanger in tumor cells.
Topics: Animals; Asparagine; Asparagus Plant; Citrus; Flavones; Humans; Mice; Microscopy, Electron
PubMed: 27203369
DOI: 10.1016/j.chembiol.2016.05.007 -
Ecotoxicology and Environmental Safety Nov 2022Increased production and environmental release of multi-walled carbon nanotubes (MWCNTs) increase soil exposure and potential risk to earthworms. However, MWCNT toxicity...
Increased production and environmental release of multi-walled carbon nanotubes (MWCNTs) increase soil exposure and potential risk to earthworms. However, MWCNT toxicity to earthworms remains unclear, with some studies identifying negative effects and others negligible effects. In this study, to determine whether exposure to MWCNTs negatively affects earthworms and to elucidate possible mechanisms of toxicity, earthworms were exposed to sublethal soil concentrations of MWCNTs (10, 50, and 100 mg/kg) for 28 days. Earthworm growth and reproduction, activities of cytochrome P450 (CYP) isoforms (1A2, 2C9, and 3A4) and antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione-s-transferase (GST)), and metabolomics were determined. Effects of MWCNTs on earthworms depended on exposure concentration. Exposure to MWCNTs did not significantly affect growth and reproduction of individual earthworms. Exposure to 50 mg/kg MWCNTs significantly increased activities of CYP2C9, CYP3A4, SOD, CAT, and GST but clearly reduced levels of L-aspartate, L-asparagine, and glutamine. With exposure to 100 mg/kg MWCNTs, toxic effects on earthworms were observed, with significant inhibition in activities of CYP isoenzymes and SOD, significant reductions in L-aspartate, L-asparagine, glutamine, and tryptophan, and simultaneous accumulations of citrate, isocitrate, fumarate, 2-oxoglutarate, pyruvate, D-galactose, carbamoyl phosphate, formyl anthranilate, hypoxanthine, and xanthine. Results suggest that toxicity of MWCNTs to earthworms is associated with reduced detoxification capacity, excessive oxidative stress, and disturbance of multiple metabolic pathways, including amino acids metabolism, the tricarboxylic acid cycle, pyruvate metabolism, D-galactose metabolism, and purine metabolism. The study provides new insights to better understand and predict the toxicity of MWCNTs in soil.
Topics: Animals; Oligochaeta; Nanotubes, Carbon; Soil; Glutamine; Galactose; Aspartic Acid; Asparagine; Oxidative Stress; Superoxide Dismutase; Soil Pollutants; Glutathione Transferase; Reproduction; Pyruvates
PubMed: 36228358
DOI: 10.1016/j.ecoenv.2022.114158 -
The Biochemical Journal Jul 1996Now that enzymes are available that are stable above 100 degrees C it is possible to investigate conformational stability at this temperature, and also the effect of... (Review)
Review
Now that enzymes are available that are stable above 100 degrees C it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 degrees C than in those stable at 50 degrees C, or that the structures of very stable proteins are systematically different from those of less stable proteins. The major degradative mechanisms are deamidation of asparagine and glutamine, and succinamide formation at aspartate and glutamate leading to peptide bond hydrolysis. In addition to being temperature-dependent, these reactions are strongly dependent upon the conformational freedom of the susceptible amino acid residues. Evidence is accumulating which suggests that even at 100 degrees C deamidation and succinamide formation proceed slowly or not at all in conformationally intact (native) enzymes. Whether this is the case at higher temperatures is not yet clear, so it is not known whether denaturation of degradation will set the upper limit of stability for enzymes.
Topics: Asparagine; Aspartic Acid; Enzymes; Hot Temperature; Models, Chemical; Protein Conformation; Protein Denaturation
PubMed: 8694749
DOI: 10.1042/bj3170001 -
Journal of Agricultural and Food... Dec 2016Acrylamide forms from free asparagine and reducing sugars during frying, baking, roasting, or high-temperature processing, and cereal products are major contributors to...
Acrylamide forms from free asparagine and reducing sugars during frying, baking, roasting, or high-temperature processing, and cereal products are major contributors to dietary acrylamide intake. Free asparagine concentration is the determining factor for acrylamide-forming potential in cereals, and this study investigated the effect of fungicide application on free asparagine accumulation in wheat grain. Free amino acid concentrations were measured in flour from 47 varieties of wheat grown in a field trial in 2011-2012. The wheat had been supplied with nitrogen and sulfur and treated with growth regulators and fungicides. Acrylamide formation was measured after the flour had been heated at 180 °C for 20 min. Flour was also analyzed from 24 (of the 47) varieties grown in adjacent plots that were treated in identical fashion except that no fungicide was applied, resulting in visible infection by Septoria tritici, yellow rust, and brown rust. Free asparagine concentration in the fungicide-treated wheat ranged from 1.596 to 3.987 mmol kg, with a significant (p < 0.001 to p = 0.006, F test) effect of variety for not only free asparagine but all of the free amino acids apart from cysteine and ornithine. There was also a significant (p < 0.001, F test) effect of variety on acrylamide formation, which ranged from 134 to 992 μg kg. There was a significant (p < 0.001, F test) correlation between free asparagine concentration and acrylamide formation. Both free asparagine concentration and acrylamide formation increased in response to a lack of fungicide treatment, the increases in acrylamide ranging from 2.7 to 370%. Free aspartic acid concentration also increased, whereas free glutamic acid concentration increased in some varieties but decreased in others, and free proline concentration decreased. The study showed disease control by fungicide application to be an important crop management measure for mitigating the problem of acrylamide formation in wheat products.
Topics: Acrylamide; Amino Acids; Asparagine; Cooking; Flour; Fungicides, Industrial; Triticum
PubMed: 27977182
DOI: 10.1021/acs.jafc.6b04520