Genetic Code Expansion: A Powerful Tool for Understanding the Physiological Consequences of Oxidative Stress Protein Modifications

Posttranslational modifications resulting from oxidation of proteins (Ox-PTMs) are present intracellularly under conditions of oxidative stress as well as basal conditions. In the past, these modifications were thought to be generic protein damage, but it has become increasingly clear that Ox-PTMs can have specific physiological effects. It is an arduous task to distinguish between the two cases, as multiple Ox-PTMs occur simultaneously on the same protein, convoluting analysis. Genetic code expansion (GCE) has emerged as a powerful tool to overcome this challenge as it allows for the site-specific incorporation of an Ox-PTM into translated protein. The resulting homogeneously modified protein products can then be rigorously characterized for the effects of individual Ox-PTMs. We outline the strengths and weaknesses of GCE as they relate to the field of oxidative stress and Ox-PTMs. An overview of the Ox-PTMs that have been genetically encoded and applications of GCE to the study of Ox-PTMs, including antibody validation and therapeutic development, is described.

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Genetically Encoded Libraries of Nonstandard Peptides

Journal of Nucleic Acids ◽

10.1155/2012/713510 ◽

2012 ◽

Vol 2012 ◽

pp. 1-15 ◽

Cited By ~ 15

Author(s):

Takashi Kawakami ◽

Hiroshi Murakami

Keyword(s):

Amino Acids ◽

Genetic Code ◽

Posttranslational Modifications ◽

Biological Properties ◽

Peptide Libraries ◽

Universal Genetic Code ◽

Nonsense Codons ◽

Genetic Code Expansion ◽

Diagnostic Applications ◽

Selection Of

The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.

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The PPAR Ω Pocket: Renewed Opportunities for Drug Development

PPAR Research ◽

10.1155/2020/9657380 ◽

2020 ◽

Vol 2020 ◽

pp. 1-21

Author(s):

Åsmund Kaupang ◽

Trond Vidar Hansen

Keyword(s):

Gene Expression ◽

Posttranslational Modifications ◽

Expression Patterns ◽

Gene Expression Patterns ◽

Therapeutic Effects ◽

Physiological Effects ◽

Binding Modes ◽

New Class ◽

The Past ◽

New Framework

The past decade of PPARγ research has dramatically improved our understanding of the structural and mechanistic bases for the diverging physiological effects of different classes of PPARγ ligands. The discoveries that lie at the heart of these developments have enabled the design of a new class of PPARγ ligands, capable of isolating central therapeutic effects of PPARγ modulation, while displaying markedly lower toxicities than previous generations of PPARγ ligands. This review examines the emerging framework around the design of these ligands and seeks to unite its principles with the development of new classes of ligands for PPARα and PPARβ/δ. The focus is on the relationships between the binding modes of ligands, their influence on PPAR posttranslational modifications, and gene expression patterns. Specifically, we encourage the design and study of ligands that primarily bind to the Ω pockets of PPARα and PPARβ/δ. In support of this development, we highlight already reported ligands that if studied in the context of this new framework may further our understanding of the gene programs regulated by PPARα and PPARβ/δ. Moreover, recently developed pharmacological tools that can be utilized in the search for ligands with new binding modes are also presented.

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Faculty Opinions recommendation of A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1057894.512855 ◽

2007 ◽

Author(s):

Niles Lehman

Keyword(s):

Genetic Code ◽

Genetic Encoding ◽

Genetic Code Expansion

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Structural Basis for Genetic-Code Expansion with Various Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase

SSRN Electronic Journal ◽

10.2139/ssrn.3244022 ◽

2018 ◽

Cited By ~ 1

Author(s):

Tatsuo Yanagisawa ◽

Mitsuo Kuratani ◽

Eiko Seki ◽

Nobumasa Hino ◽

Kensaku Sakamoto ◽

...

Keyword(s):

Genetic Code ◽

Trna Synthetase ◽

Structural Basis ◽

Genetic Code Expansion

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Cytokinin N-glucosides: Occurrence, Metabolism and Biological Activities in Plants

Biomolecules ◽

10.3390/biom11010024 ◽

2020 ◽

Vol 11 (1) ◽

pp. 24

Author(s):

Eva Pokorná ◽

Tomáš Hluska ◽

Petr Galuszka ◽

H. Tucker Hallmark ◽

Petre I. Dobrev ◽

...

Keyword(s):

Metabolic Pathways ◽

Growth And Development ◽

Biological Activities ◽

Physiological Effects ◽

Inhibitory Effects ◽

Plant Growth And Development ◽

The Past ◽

Complex Process ◽

Considerable Impact ◽

Physiological Impact

Cytokinins (CKs) are a class of phytohormones affecting many aspects of plant growth and development. In the complex process of CK homeostasis in plants, N-glucosylation represents one of the essential metabolic pathways. Its products, CK N7- and N9-glucosides, have been largely overlooked in the past as irreversible and inactive CK products lacking any relevant physiological impact. In this work, we report a widespread distribution of CK N-glucosides across the plant kingdom proceeding from evolutionary older to younger plants with different proportions between N7- and N9-glucosides in the total CK pool. We show dramatic changes in their profiles as well as in expression levels of the UGT76C1 and UGT76C2 genes during Arabidopsis ontogenesis. We also demonstrate specific physiological effects of CK N-glucosides in CK bioassays including their antisenescent activities, inhibitory effects on root development, and activation of the CK signaling pathway visualized by the CK-responsive YFP reporter line, TCSv2::3XVENUS. Last but not least, we present the considerable impact of CK N7- and N9-glucosides on the expression of CK-related genes in maize and their stimulatory effects on CK oxidase/dehydrogenase activity in oats. Our findings revise the apparent irreversibility and inactivity of CK N7- and N9-glucosides and indicate their involvement in CK evolution while suggesting their unique function(s) in plants.

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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Increased oxidative stress and plasma Hsp70 levels among gasoline filling station attendants

Toxicology and Industrial Health ◽

10.1177/0748233715616554 ◽

2016 ◽

Vol 33 (2) ◽

pp. 171-181 ◽

Cited By ~ 14

Author(s):

Bing Xia ◽

Kangcheng Chen ◽

Yingnan Lv ◽

Damin Huang ◽

Jing Liu ◽

...

Keyword(s):

Oxidative Stress ◽

Stress Protein ◽

Weighted Average ◽

Exposed Group ◽

Cumulative Exposure ◽

Enzyme Linked Immunosorbent Assay ◽

Control Group ◽

Gasoline Station ◽

Filling Station ◽

Exposed Workers

Objectives: Methylcyclopentadienyl manganese tricarbonyl (MMT) is an organic derivative of manganese (Mn) and is used as an antiknock agent and octane enhancer in gasoline. In this article, we tested the oxidative stress and heat stress protein (Hsp) 70 levels of gasoline station attendants to explore potential plasma biomarkers. Furthermore, the dose–response relationship was also identified. Methods: A total of 144 workers, including 96 petrol fillers and 48 cashiers, participated in the study. Ambient concentrations of benzene, toluene, ethylbenzene, and xylene (BTEX) and Mn were monitored at nine filling stations. During the measuring process, the individual cumulative exposure index was calculated. Plasma oxidative stress and Hsp70 levels were also analysed using enzyme-linked immunosorbent assay. Results: The BTEX time-weighted average in office areas was significantly lower than in refuelling areas ( p < 0.05). In refuelling areas, the content of Mn ranged from 6.44 μg/m3 to 127.34 μg/m3, which was much higher than that in office areas (3.16–7.22 μg/m3; p < 0.05). Exposed workers had significantly different plasma oxidative stress indicators compared with the control group, respectively: superoxide dismutase (SOD), 39.18 ± 6.05 U/mL versus 52.84 ± 3.87 U/mL; glutathione peroxidase (GSH-Px), 186.07 ± 15.63 U versus 194.38 ± 10.42 U; and malondialdehyde (MDA), 1.68 ± 0.52 nmol/L versus 1.43 ± 0.64 nmol/L (in all comparisons, p < 0.05). Plasma Hsp70 level in the exposed group (2.77 ± 0.64 ng/mL) was significantly higher than in the control group (2.32 ± 0.87 ng/mL; p < 0.05). Furthermore, Hsp70 levels were inversely correlated with the activities of SOD ( r = −0.305) and GSH-Px ( r = −0.302) in the exposed group ( p < 0.05). Moreover, a positive correlation ( r = 0.653) was found between plasma Hsp70 levels and plasma MDA levels ( p < 0.05). Conclusion: Exposure to MMT-containing gasoline may result in increasing reactive oxygen stress among filling station attendants. Plasma Hsp70 levels could be used as a sensitive responsive biomarker for exposed workers.

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