scholarly journals Genetic Incorporation of Two Mutually Orthogonal Bioorthogonal Amino Acids That Enable Efficient Protein Dual-Labeling in Cells

2021 ◽  
Author(s):  
Riley M. Bednar ◽  
Subhashis Jana ◽  
Sahiti Kuppa ◽  
Rachel Franklin ◽  
Joseph Beckman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genetic Incorporation ◽  
In Cells

Two site genetic incorporation of varying length polyethylene glycol into the backbone of one peptide

2015 ◽  
Vol 51 (76) ◽  
pp. 14385-14388 ◽  
Author(s):  
Qingmin Zang ◽  
Seiichi Tada ◽  
Takanori Uzawa ◽  
Daisuke Kiga ◽  
Masayuki Yamamura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Polyethylene Glycol ◽  
Varying Length ◽  
Genetic Incorporation

Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of a polypeptide using stop-anticodon and frameshift anticodon-containing tRNAs, which were acylated with PEG-containing amino acids.

scholarly journals Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Howard Gamper ◽  
Haixing Li ◽  
Isao Masuda ◽  
D. Miklos Robkis ◽  
Thomas Christian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Conformational Change ◽  
Molecular Mechanism ◽  
Late Stage ◽  
Anticodon Loop ◽  
Pairing Mechanism ◽  
Extra Nucleotide ◽  
In Cells

AbstractWhile genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

Transport of L-tyrosine by B16/F10 melanoma cells: the effect of the intracellular content of other amino acids

1990 ◽  
Vol 97 (3) ◽  
pp. 479-485
Author(s):  
J.R. Jara ◽  
J.H. Martinez-Liarte ◽  
F. Solano ◽  
R. Penafiel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Initial Rate ◽  
Aromatic Amino Acids ◽  
Melanoma Cells ◽  
Transport Systems ◽  
Specific Amino Acid ◽  
Intracellular Content ◽  
Tyrosine Hydroxylation ◽  
In Cells

The uptake of L-Tyr by B16/F10 malignant melanocytes in culture has been studied. These melanoma cells can either be depleted of amino acids by 1 h preincubation in Hanks' isotonic medium or preloaded with a specific amino acid by 1 h preincubation in the same solution containing 2 mM of the amino acid to be preloaded. By means of these pretreatments, it is shown that the rate of L-Tyr uptake is greatly dependent on the content of other amino acids inside the cells. The L-Tyr uptake is higher in cells preloaded with amino acids transported by the L and ASC systems than in cells depleted of amino acids or preloaded with amino acids transported by the A system. It is concluded that L-Tyr is mainly taken up by an exchange mechanism with other amino acids mediated by the L1 system, although the ASC system can also participate in the process. In agreement with that, the homo-exchange performed by cells preloaded with unlabelled L-Tyr is more efficient than any other hetero-exchange, although L-Dopa, the product of tyrosine hydroxylation in melanin synthesis, is almost as efficient as L-Tyr. Apart from aromatic amino acids, melanoma cells preloaded with L-Met and L-His also yield a high initial rate of L-Tyr uptake. The results herein suggest that melanoma cells do not have transport systems specific for L-Tyr, even if this amino acid is needed to carry out the differential pathway of this type of cells, melanosynthesis.

scholarly journals ATF4-Mediated Upregulation of REDD1 and Sestrin2 Suppresses mTORC1 Activity during Prolonged Leucine Deprivation

2019 ◽  
Vol 150 (5) ◽  
pp. 1022-1030 ◽  
Author(s):  
Dandan Xu ◽  
Weiwei Dai ◽  
Lydia Kutzler ◽  
Holly A Lacko ◽  
Leonard S Jefferson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dna Damage ◽  
Amino Acid ◽  
Protein Kinase ◽  
Dna Damage Response ◽  
Dependent Manner ◽  
Mouse Embryo Fibroblasts ◽  
Damage Response ◽  
Mtorc1 Activation ◽  
In Cells

ABSTRACT Background The protein kinase target of rapamycin (mTOR) in complex 1 (mTORC1) is activated by amino acids and in turn upregulates anabolic processes. Under nutrient-deficient conditions, e.g., amino acid insufficiency, mTORC1 activity is suppressed and autophagy is activated. Intralysosomal amino acids generated by autophagy reactivate mTORC1. However, sustained mTORC1 activation during periods of nutrient insufficiency would likely be detrimental to cellular homeostasis. Thus, mechanisms must exist to prevent amino acids released by autophagy from reactivating the kinase. Objective The objective of the present study was to test whether mTORC1 activity is inhibited during prolonged leucine deprivation through ATF4-dependent upregulation of the mTORC1 suppressors regulated in development and DNA damage response 1 (REDD1) and Sestrin2. Methods Mice (8 wk old; C57Bl/6 × 129SvEV) were food deprived (FD) overnight and one-half were refed the next morning. Mouse embryo fibroblasts (MEFs) deficient in ATF4, REDD1, and/or Sestrin2 were deprived of leucine for 0–16 h. mTORC1 activity and ATF4, REDD1, and Sestrin2 expression were assessed in liver and cell lysates. Results Refeeding FD mice resulted in activation of mTORC1 in association with suppressed expression of both REDD1 and Sestrin2 in the liver. In cells in culture, mTORC1 exhibited a triphasic response to leucine deprivation, with an initial suppression followed by a transient reactivation from 2 to 4 h and a subsequent resuppression after 8 h. Resuppression occurred concomitantly with upregulated expression of ATF4, REDD1, and Sestrin2. However, in cells lacking ATF4, neither REDD1 nor Sestrin2 expression was upregulated by leucine deprivation, and resuppression of mTORC1 was absent. Moreover, in cells lacking either REDD1 or Sestrin2, mTORC1 resuppression was attenuated, and in cells lacking both proteins resuppression was further blunted. Conclusions The results suggest that leucine deprivation upregulates expression of both REDD1 and Sestrin2 in an ATF4-dependent manner, and that upregulated expression of both proteins is involved in resuppression of mTORC1 during prolonged leucine deprivation.

scholarly journals Genetic Incorporation of Noncanonical Amino Acids Using Two Mutually Orthogonal Quadruplet Codons

2019 ◽  
Vol 8 (5) ◽  
pp. 1168-1174 ◽  
Author(s):  
Erome Daniel Hankore ◽  
Linyi Zhang ◽  
Yan Chen ◽  
Kun Liu ◽  
Wei Niu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Noncanonical Amino Acids ◽  
Genetic Incorporation

scholarly journals Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Maren Kirstin Schuhmacher ◽  
Serap Beldar ◽  
Mina S. Khella ◽  
Alexander Bröhm ◽  
Jan Ludwig ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Histone H3 ◽  
Protein Methylation ◽  
Sequence Specificity ◽  
Sequence Design ◽  
Substrate Peptide ◽  
Lysine Methyltransferase ◽  
In Cells

AbstractSETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.

scholarly journals Regulation of rRNA synthesis and processing in animal cells. Effect of nucleoside analogues

1982 ◽  
Vol 202 (2) ◽  
pp. 325-332 ◽  
Author(s):  
S Iapalucci-Espinoza ◽  
M T Franze-Fernandez
Keyword(s):  
Amino Acids ◽  
High Rate ◽  
Nucleoside Analogues ◽  
Rrna Synthesis ◽  
Complete Medium ◽  
Animal Cells ◽  
Rrna Transcription ◽  
Ehrlich Ascites ◽  
Ehrlich Ascites Cells ◽  
In Cells

The nucleoside analogues fluorouridine and fluorodeoxyuridine (both at 100 muM) and 8-azaguanine (at 500 muM) inhibit both rRNA transcription and processing in Ehrlich ascites cells. In BHK21 cells fluorodeoxyuridine has no effect on either rRNA maturation or transcription, whereas toyocamycin (at 2 microM) inhibits both processes in BHK21 cells and Ehrlich ascites cells. The drugs inhibit transcription in cells incubated in the complete medium, but have no effect on the decreased transcription in cells incubated in a medium without amino acids. This lack of effect cannot be explained by an altered uptake of the drugs in the amino acid-starved cells, since maturation of the rRNA precursor is affected in cells incubated in media with or without amino acids. The effect of the drugs on rRNA transcription is not the consequence of the inhibition of protein synthesis. The results lend support to the proposal that rRNA processing and transcription are co-ordinately controlled in cells with a high rate of rRNA synthesis.

scholarly journals Characterization of Dengue Virus NS4A and NS4B Protein Interaction

2015 ◽  
Vol 89 (7) ◽  
pp. 3455-3470 ◽  
Author(s):  
Jing Zou ◽  
Xuping Xie ◽  
Qing-Yin Wang ◽  
Hongping Dong ◽  
Michelle Yueqi Lee ◽  
...  
Keyword(s):  
Amino Acids ◽  
Dengue Virus ◽  
Viral Replication ◽  
Transmembrane Domain ◽  
Nmr Analysis ◽  
Replication Complex ◽  
Essential Components ◽  
Infected Cells ◽  
Er Membrane ◽  
In Cells

ABSTRACTFlavivirus replication is mediated by a membrane-associated replication complex where viral membrane proteins NS2A, NS2B, NS4A, and NS4B serve as the scaffold for the replication complex formation. Here, we used dengue virus serotype 2 (DENV-2) as a model to characterize viral NS4A-NS4B interaction. NS4A interacts with NS4B in virus-infected cells and in cells transiently expressing NS4A and NS4B in the absence of other viral proteins. Recombinant NS4A and NS4B proteins directly bind to each other with an estimatedKd(dissociation constant) of 50 nM. Amino acids 40 to 76 (spanning the first transmembrane domain, consisting of amino acids 50 to 73) of NS4A and amino acids 84 to 146 (also spanning the first transmembrane domain, consisting of amino acids 101 to 129) of NS4B are the determinants for NS4A-NS4B interaction. Nuclear magnetic resonance (NMR) analysis suggests that NS4A residues 17 to 80 form two amphipathic helices (helix α1, comprised of residues 17 to 32, and helix α2, comprised of residues 40 to 47) that associate with the cytosolic side of endoplasmic reticulum (ER) membrane and helix α3 (residues 52 to 75) that transverses the ER membrane. In addition, NMR analysis identified NS4A residues that may participate in the NS4A-NS4B interaction. Amino acid substitution of these NS4A residues exhibited distinct effects on viral replication. Three of the four NS4A mutations (L48A, T54A, and L60A) that affected the NS4A-NS4B interaction abolished or severely reduced viral replication; in contrast, two NS4A mutations (F71A and G75A) that did not affect NS4A-NS4B interaction had marginal effects on viral replication, demonstrating the biological relevance of the NS4A-NS4B interaction to DENV-2 replication. Taken together, the study has provided experimental evidence to argue that blocking the NS4A-NS4B interaction could be a potential antiviral approach.IMPORTANCEFlavivirus NS4A and NS4B proteins are essential components of the ER membrane-associated replication complex. The current study systematically characterizes the interaction between flavivirus NS4A and NS4B. Using DENV-2 as a model, we show that NS4A interacts with NS4B in virus-infected cells, in cells transiently expressing NS4A and NS4B proteins, orin vitrowith recombinant NS4A and NS4B proteins. We mapped the minimal regions required for the NS4A-NS4B interaction to be amino acids 40 to 76 of NS4A and amino acids 84 to 146 of NS4B. NMR analysis revealed the secondary structure of amino acids 17 to 80 of NS4A and the NS4A amino acids that may participate in the NS4A-NS4B interaction. Functional analysis showed a correlation between viral replication and NS4A-NS4B interaction, demonstrating the biological importance of the NS4A-NS4B interaction. The study has advanced our knowledge of the molecular function of flavivirus NS4A and NS4B proteins. The results also suggest that inhibitors of the NS4A-NS4B interaction could be pursued for flavivirus antiviral development.

scholarly journals Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

1989 ◽  
Vol 9 (6) ◽  
pp. 2706-2714 ◽  
Author(s):  
A Cooper ◽  
H Bussey
Keyword(s):  
Amino Acids ◽  
Gene Product ◽  
Killer Toxin ◽  
Secreted Protein ◽  
Serine Carboxypeptidase ◽  
Carboxypeptidase B ◽  
Basic Amino Acids ◽  
Precursor Proteins ◽  
In Cells

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.

Genetic Incorporation of Multiple Unnatural Amino Acids into Proteins in Mammalian Cells

2013 ◽  
Vol 125 (52) ◽  
pp. 14330-14333 ◽  
Author(s):  
Han Xiao ◽  
Abhishek Chatterjee ◽  
Sei-hyun Choi ◽  
Krishna M. Bajjuri ◽  
Subhash C. Sinha ◽  
...  
Keyword(s):  
Amino Acids ◽  
Unnatural Amino Acids ◽  
Mammalian Cells ◽  
Genetic Incorporation
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