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The Plant Journal : For Cell and... Nov 1991
Review
Topics: Asparagine; Aspartate Aminotransferases; Aspartate-Ammonia Ligase; Gene Expression Regulation; Genes, Plant; Glutamate-Ammonia Ligase; Glutamine; Isoenzymes; Multigene Family; Plants
PubMed: 1688250
DOI: 10.1046/j.1365-313x.1991.00999.x -
The FEBS Journal Mar 2018C-mannosylation is a rare type of protein glycosylation, the functions and mechanisms of which remain unclear. Recently, we identified DPY19L3 as a C-mannosyltransferase...
C-mannosylation is a rare type of protein glycosylation, the functions and mechanisms of which remain unclear. Recently, we identified DPY19L3 as a C-mannosyltransferase of R-spondin1 in human cells. DPY19L3 is predicted to be a multipass transmembrane protein that localizes to the endoplasmic reticulum (ER); however, its structure is undetermined. In this study, we propose a topological structure of DPY19L3 by in silico analysis and experimental methods such as redox-sensitive luciferase assay and introduction of N-glycosylation sites, suggesting that DPY19L3 comprises 11 transmembrane regions and two re-entrant loops with the N- and C-terminal ends facing the cytoplasm and ER lumen, respectively. Furthermore, DPY19L3 has four predicted N-glycosylation sites, and we have demonstrated that DPY19L3 is N-glycosylated at Asn and Asn but not Asn and Asn , supporting our topological model. By mass spectrometry, we measured the C-mannosyltransferase activity of N-glycosylation-defective mutants of DPY19L3 and isoform2, a splice variant, which lacks the C-terminal luminal region of DPY19L3. Isoform2 does not possess C-mannosyltransferase activity, indicating the importance of the C-terminal region; however, N-glycosylations of DPY19L3 do not have any roles for its enzymatic activity. These novel findings on DPY19L3 provide important insights into the mechanism of C-mannosylation.
Topics: Amino Acid Sequence; Asparagine; Binding Sites; Computer Simulation; Endoplasmic Reticulum; Glycosylation; HEK293 Cells; Humans; Mannose; Mannosyltransferases; Membrane Proteins; Mutation; Protein Domains; Sequence Homology, Amino Acid
PubMed: 29405629
DOI: 10.1111/febs.14398 -
European Journal of Biochemistry Mar 2003To sustain life mammals have an absolute and continual requirement for oxygen, which is necessary to produce energy for normal cell survival and growth. Hence,... (Review)
Review
To sustain life mammals have an absolute and continual requirement for oxygen, which is necessary to produce energy for normal cell survival and growth. Hence, maintaining oxygen homeostasis is a critical requirement and mammals have evolved a wide range of cellular and physiological responses to adapt to changes in oxygen availability. In the past few years it has become evident that the transcriptional protein complex hypoxia-inducible factor (HIF) is a key regulator of these processes. In this review we will focus on the way oxygen availability regulates HIF proteins and in particular we will discuss the way oxygen-dependent hydroxylation of specific amino acid residues has been demonstrated to regulate HIF function at the level of both protein stability and transcriptional potency.
Topics: Asparagine; Hydroxylation; Hypoxia; Oxygen; Proline; Transcription Factors
PubMed: 12603311
DOI: 10.1046/j.1432-1033.2003.03445.x -
Biochemistry May 2023In photosynthetic reaction centers from purple bacteria (PbRCs), light-induced charge separation leads to the reduction of the terminal electron acceptor quinone, Q. The...
In photosynthetic reaction centers from purple bacteria (PbRCs), light-induced charge separation leads to the reduction of the terminal electron acceptor quinone, Q. The reduction of Q to Q is followed by protonation via Asp-L213 and Ser-L223 in PbRC from . However, Asp-L213 is replaced with nontitratable Asn-L222 and Asn-L213 in PbRCs from and , respectively. Here, we investigated the energetics of proton transfer along the asparagine-involved H-bond network using a quantum mechanical/molecular mechanical approach. The potential energy profile for the H-bond between HO and the carbonyl O site of Asn-L222 shows that the proton is predominantly localized at the Asn-L222 moiety in the PbRC protein environment, easily forming the enol species. The release of the proton from the amide -NH site toward Ser-L232 via tautomerization suffers from the energy barrier. Upon reorientation of Asn-L222, the enol -OH site forms a short low-barrier H-bond with Ser-L232, facilitating protonation of Q in a Grotthuss-like mechanism. This is a basis of how asparagine or glutamine side chains function as acceptors/donors in proton transfer pathways.
Topics: Protons; Electron Transport; Oxidation-Reduction; Asparagine; Photosynthetic Reaction Center Complex Proteins; Mutagenesis, Site-Directed; Rhodobacter sphaeroides; Kinetics
PubMed: 37083399
DOI: 10.1021/acs.biochem.3c00013 -
PLoS Computational Biology Apr 2017Many prion-forming proteins contain glutamine/asparagine (Q/N) rich domains, and there are conflicting opinions as to the role of primary sequence in their conversion to...
Many prion-forming proteins contain glutamine/asparagine (Q/N) rich domains, and there are conflicting opinions as to the role of primary sequence in their conversion to the prion form: is this phenomenon driven primarily by amino acid composition, or, as a recent computational analysis suggested, dependent on the presence of short sequence elements with high amyloid-forming potential. The argument for the importance of short sequence elements hinged on the relatively-high accuracy obtained using a method that utilizes a collection of length-six sequence elements with known amyloid-forming potential. We weigh in on this question and demonstrate that when those sequence elements are permuted, even higher accuracy is obtained; we also propose a novel multiple-instance machine learning method that uses sequence composition alone, and achieves better accuracy than all existing prion prediction approaches. While we expect there to be elements of primary sequence that affect the process, our experiments suggest that sequence composition alone is sufficient for predicting protein sequences that are likely to form prions. A web-server for the proposed method is available at http://faculty.pieas.edu.pk/fayyaz/prank.html, and the code for reproducing our experiments is available at http://doi.org/10.5281/zenodo.167136.
Topics: Amino Acid Sequence; Amyloid; Asparagine; Computational Biology; Glutamine; Humans; Machine Learning; Prions; Yeasts
PubMed: 28394888
DOI: 10.1371/journal.pcbi.1005465 -
Journal of Biomolecular NMR Jan 2021The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine...
The monitoring of non-enzymatic post-translational modifications (PTMs) in therapeutic proteins is important to ensure drug safety and efficacy. Together with methionine and asparagine, aspartic acid (Asp) is very sensitive to spontaneous alterations. In particular, Asp residues can undergo isomerization and peptide-bond hydrolysis, especially when embedded in sequence motifs that are prone to succinimide formation or when followed by proline (Pro). As Asp and isoAsp have the same mass, and the Asp-Pro peptide-bond cleavage may lead to an unspecific mass difference of + 18 Da under native conditions or in the case of disulfide-bridged cleavage products, it is challenging to directly detect and characterize such modifications by mass spectrometry (MS). Here we propose a 2D NMR-based approach for the unambiguous identification of isoAsp and the products of Asp-Pro peptide-bond cleavage, namely N-terminal Pro and C-terminal Asp, and demonstrate its applicability to proteins including a therapeutic monoclonal antibody (mAb). To choose the ideal pH conditions under which the NMR signals of isoAsp and C-terminal Asp are distinct from other random coil signals, we determined the pK values of isoAsp and C-terminal Asp in short peptides. The characteristic H-C chemical shift correlations of isoAsp, N-terminal Pro and C-terminal Asp under standardized conditions were used to identify these PTMs in lysozyme and in the therapeutic mAb rituximab (MabThera) upon prolonged storage under acidic conditions (pH 4-5) and 40 °C. The results show that the application of our 2D NMR-based protocol is straightforward and allows detecting chemical changes of proteins that may be otherwise unnoticed with other analytical methods.
Topics: Amino Acid Sequence; Asparagine; Aspartic Acid; Isomerism; Magnetic Resonance Spectroscopy; Nuclear Magnetic Resonance, Biomolecular; Peptides; Proteins; Structure-Activity Relationship
PubMed: 33475951
DOI: 10.1007/s10858-020-00356-4 -
Biochemistry Dec 2017Coiled coils (CCs) are among the best-understood protein folds. Nonetheless, there are gaps in our knowledge of CCs. Notably, CCs are likely to be structurally more...
Coiled coils (CCs) are among the best-understood protein folds. Nonetheless, there are gaps in our knowledge of CCs. Notably, CCs are likely to be structurally more dynamic than often considered. Here, we explore this in an abundant class of CCs, parallel dimers, focusing on polar asparagine (Asn) residues in the hydrophobic interface. It is well documented that such inclusions discriminate between different CC oligomers, which has been rationalized in terms of whether the Asn can make side-chain hydrogen bonds. Analysis of parallel CC dimers in the Protein Data Bank reveals a variety of Asn side-chain conformations, but not all of these make the expected inter-side-chain hydrogen bond. We probe the structure and dynamics of a de novo-designed coiled-coil homodimer, CC-Di, by multidimensional nuclear magnetic resonance spectroscopy, including model-free dynamical analysis and relaxation-dispersion experiments. We find dynamic exchange on the millisecond time scale between Asn conformers with the side chains pointing into and out of the core. We perform molecular dynamics simulations that are consistent with this, revealing that the side chains are highly dynamic, exchanging between hydrogen-bonded-paired conformations in picoseconds to nanoseconds. Combined, our data present a more dynamic view for Asn at CC interfaces. Although inter-side-chain hydrogen bonding states are the most abundant, Asn is not always buried or engaged in such interactions. Because interfacial Asn residues are key design features for modulating CC stability and recognition, these further insights into how they are accommodated within CC structures will aid their predictive modeling, engineering, and design.
Topics: Amino Acid Sequence; Asparagine; Computer Simulation; Databases, Protein; Dimerization; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Protein Conformation; Protein Structure, Secondary; Proteins
PubMed: 29166010
DOI: 10.1021/acs.biochem.7b00848 -
Virology Apr 2016Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein...
Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesis of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine.
Topics: Amino Acid Motifs; Animals; Asparagine; Crystallization; Equine Infectious Anemia; Horses; Hydrogen Bonding; Infectious Anemia Virus, Equine; Peptides; Protein Structure, Secondary; Viral Envelope Proteins
PubMed: 26874586
DOI: 10.1016/j.virol.2016.01.010 -
Amino Acids Sep 2016Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging...
Racemization in proteins and peptides at sites of L-asparaginyl and L-aspartyl residues contributes to their spontaneous degradation, especially in the biological aging process. Amino acid racemization involves deprotonation of the alpha carbon and replacement of the proton in the opposite stereoconfiguration; this reaction is much faster for aspartate/asparagine than for other amino acids because these residues form a succinimide ring in which resonance stabilizes the carbanion resulting from proton loss. To determine if the replacement of the hydrogen atom on the alpha carbon with a deuterium atom might decrease the rate of racemization and thus stabilize polypeptides, we synthesized a hexapeptide, VYPNGA, in which the three carbon-bound protons in the asparaginyl residue were replaced with deuterium atoms. Upon incubation of this peptide in pH 7.4 buffer at 37 °C, we found that the rate of deamidation via the succinimide intermediate was unchanged by the presence of the deuterium atoms. However, the accumulation of the D-aspartyl and D-isoaspartyl-forms resulting from racemization and hydrolysis of the succinimide was decreased more than five-fold in the deuterated peptide over a 20 day incubation at physiological temperature and pH. Additionally, we found that the succinimide intermediate arising from the degradation of the deuterated asparaginyl peptide was slightly less likely to open to the isoaspartyl configuration than was the protonated succinimide. These findings suggest that the kinetic isotope effect resulting from the presence of deuteriums in asparagine residues can limit the accumulation of at least some of the degradation products that arise as peptides and proteins age.
Topics: Asparagine; Deuterium; Oligopeptides
PubMed: 27169868
DOI: 10.1007/s00726-016-2250-z -
Journal of Virology Jul 2019Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype...
Viruses actively interact with host metabolism because viral replication relies on host cells to provide nutrients and energy. Vaccinia virus (VACV; the prototype poxvirus) prefers glutamine to glucose for efficient replication to the extent that VACV replication is hindered in glutamine-free medium. Remarkably, our data show that VACV replication can be fully rescued from glutamine depletion by asparagine supplementation. By global metabolic profiling, as well as genetic and chemical manipulation of the asparagine supply, we provide evidence demonstrating that the production of asparagine, which exclusively requires glutamine for biosynthesis, accounts for VACV's preference of glutamine to glucose rather than glutamine's superiority over glucose in feeding the tricarboxylic acid (TCA) cycle. Furthermore, we show that sufficient asparagine supply is required for efficient VACV protein synthesis. Our study highlights that the asparagine supply, the regulation of which has been evolutionarily tailored in mammalian cells, presents a critical barrier to VACV replication due to a high asparagine content of viral proteins and a rapid demand of viral protein synthesis. The identification of asparagine availability as a critical limiting factor for efficient VACV replication suggests a new direction of antiviral strategy development. Viruses rely on their infected host cells to provide nutrients and energy for replication. Vaccinia virus, the prototypic member of the poxviruses, which comprise many significant human and animal pathogens, prefers glutamine to glucose for efficient replication. Here, we show that the preference is not because glutamine is superior to glucose as the carbon source to fuel the tricarboxylic acid cycle for vaccinia virus replication. Rather interestingly, the preference is because the asparagine supply for efficient viral protein synthesis becomes limited in the absence of glutamine, which is necessary for asparagine biosynthesis. We provide further genetic and chemical evidence to demonstrate that asparagine availability plays a critical role in efficient vaccinia virus replication. This discovery identifies a weakness of vaccinia virus and suggests a possible direction to intervene in poxvirus infection.
Topics: Animals; Antiviral Agents; Asparagine; Cell Line; Glutamine; Humans; Metabolome; Poxviridae; Protein Biosynthesis; Vaccinia; Vaccinia virus; Viral Proteins; Virus Replication
PubMed: 30996100
DOI: 10.1128/JVI.01834-18