Genetically Encoding <i>N</i><sup>ϵ</sup>-Methyl-<scp>l</scp>-lysine in Recombinant Histones

Nguyen, Duy; Alai, Maria M. Garcia; Kapadnis, Prashant B.; Neumann, Heinz; Chin, Jason W.

doi:10.1021/ja906603s

Cited by 160 publications

(143 citation statements)

References 21 publications

(32 reference statements)

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“…This history of evolutionary pressure along with the observation that even the unmodified pyrrolysyl-tRNA synthetase PylS is relatively tolerant of nonpyrrolysine substrates [but generally utilizes them with lower efficiency (35)(36)(37)(38)(39)(40)(41)(42)], makes it an attractive system for the incorporation of unnatural amino acids either using wild-type (35,(38)(39)(40)(41)(42) or mutant pyrrolysyltRNA synthetases (35,39,(43)(44)(45)(46). Pcl mutagenesis differs from the unnatural amino acid technology in that a standard metabolite, D-Orn, is processed by the biosynthesis enzymes of a natural amino acid into a close analog of Pyl.…”

Section: Discussionmentioning

confidence: 99%

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Uno

Chiu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.protein engineering | protein medicinal chemistry | desmethylpyrrolysine | amber suppression

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Section: Discussionmentioning

confidence: 99%

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Uno

Chiu

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…This system was also employed for the synthesis of monomethyl-and dimethyl-lysine containing histones. 23,24 Schultz et al recently described the synthesis of histone H2B containing e-N-crotonyllysine at position K11 using an evolved pyrrolysyl-tRNA synthetase from Methanosarcina barkeri. 25 Here we show that the pyrrolysine system can be used to insert e-N-propionyl-(Kpr, 1), e-N-butyryl-(Kbu, 2), and e-Ncrotonyl-lysine (Kcr, 3) into histones at critical positions such as H3 K9 using the wild type pyrrolysyl-tRNA synthetase.…”

mentioning

confidence: 99%

Synthesis of ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine containing histone H3 using the pyrrolysine system

2013

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Recently new lysine modifications were detected in histones and other proteins. Using the pyrrolysine amber suppression system we genetically inserted three of the new amino acids e-N-propionyl-, e-N-butyryl-, and e-N-crotonyl-lysine site specifically into histone H3. The lysine at position 9 (H3 K9), which is known to be highly modified in chromatin, was replaced by these unnatural amino acids.The histone code is based on the post-translational modification of critical amino acid residues in different histones. Among these, lysine acetylations and methylations are the most abundant and affect the transcriptional status of the genes associated with the corresponding histones. [1][2][3] The role of these modifications is sequence dependent. Typically acetylation is associated with transcriptionally active genes, while methylation induces transcriptional silencing. 4,5 Recently Zhao and co-workers discovered a number of new modified amino acids in histones. [6][7][8] Some of these were also detected in proteins other than histones, raising the possibility that they are of more widespread importance. [9][10][11] The new post-translational modifications (PTMs) are acylated derivatives of lysine at the e-amino position. The acylation partners are propionic acid, butyric acid, malonic acid, succinic acid, crotonic acid or fatty acids. A common characteristic of these compounds is that they are key metabolic intermediates that typically exist as CoA activated thioester species in the cell. 12 It is currently not clear how these newly discovered PTMs are biosynthetically established within the histones and we do not fully understand if and how they influence genetic processes.So far no specific deacylases for e-N-propionyl-or e-Nbutyryl-lysine have been identified. However Sirt5, a member of NAD-dependent sirtuins is able to specifically deacylate e-Nmalonyl-and e-N-succinyllysine. 11 Recently it was discovered that HDAC3 exhibits decrotonylase activity in vitro 13 and it was found that lysine crotonylation activates genes even in a globally repressive environment. 7,14 In order to enable investigation of the new lysine derivatives in histones it is essential to generate histones that contain these amino acids site specifically. 15 In this direction semi-synthetic chemical ligation based methods 16,17 were utilized and chemical methods were employed to generate acetyllysine (Kac) and methylated-lysines or derivatives thereof. [18][19][20][21] Chin and co-workers reported the introduction of Kac into H3 K56 22 using the pyrrolysine system. This system was also employed for the synthesis of monomethyl-and dimethyl-lysine containing histones. 23,24 Schultz et al. recently described the synthesis of histone H2B containing e-N-crotonyllysine at position K11 using an evolved pyrrolysyl-tRNA synthetase from Methanosarcina barkeri. 25 Here we show that the pyrrolysine system can be used to insert e-N-propionyl-(Kpr, 1), e-N-butyryl-(Kbu, 2), and e-Ncrotonyl-lysine (Kcr, 3) into histones at critical positions such as H3 K9 using...

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“…Ideally, orthologous systems will be developed to allow the expression of exogenous proteins with defined, site-specific PTMs in mammalian cells but this would appear highly unrealistic, since any exogenous protein would need to be synthesized using the endogenous translational machinery. However, combinations of (1) the directed evolution of aminoacyl tRNA synthetases to utilize unnatural amino acids and/or unnatural tRNAs [74][75][76][77], (2) the further development and directed evolution of the orthologous tethered ribosome (Ribo-T) [78] such that it can read quadruplet (or greater) codons [79] and (3) development of an orthologous system for site-specific (quadruplet or greater) codon recoding [similar to the selenocysteine insertion sequence (SECIS) but for recoding only by orthologous ribosomes; [74] and (4) the use of CRISPR-type genome editing to either render endogenous genes amenable to the orthologous apparatus or to insert orthologous decoding-ready exogenous genes; may go some way to enabling the systematic study of the regulation conferred by the vast quantities of novel PTMs currently being identified.…”

Section: Realizing the Potential: Emerging Areas Technological Develmentioning

confidence: 99%

Modulation of the cytoplasmic functions of mammalian post-transcriptional regulatory proteins by methylation and acetylation: a key layer of regulation waiting to be uncovered?

Blee

Gray

Brook

2015

Biochemical Society Transactions

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Post-transcriptional control of gene expression is critical for normal cellular function and viability and many of the proteins that mediate post-transcriptional control are themselves subject to regulation by posttranslational modification (PTM), e.g. phosphorylation. However, proteome-wide studies are revealing new complexities in the PTM status of mammalian proteins, in particular large numbers of novel methylated and acetylated residues are being identified. Here we review studied examples of methylation/acetylationdependent regulation of post-transcriptional regulatory protein (PTRP) function and present collated PTM data that points to the huge potential for regulation of mRNA fate by these PTMs.

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Genetically Encoding N^ϵ-Methyl-l-lysine in Recombinant Histones

Cited by 160 publications

References 21 publications

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Synthesis of ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine containing histone H3 using the pyrrolysine system

Modulation of the cytoplasmic functions of mammalian post-transcriptional regulatory proteins by methylation and acetylation: a key layer of regulation waiting to be uncovered?

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Genetically Encoding Nϵ-Methyl-l-lysine in Recombinant Histones

Cited by 160 publications

References 21 publications

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Site-specific protein modifications through pyrroline-carboxy-lysine residues

Synthesis of ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine containing histone H3 using the pyrrolysine system

Modulation of the cytoplasmic functions of mammalian post-transcriptional regulatory proteins by methylation and acetylation: a key layer of regulation waiting to be uncovered?

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Genetically Encoding N^ϵ-Methyl-l-lysine in Recombinant Histones