Genetically Encoded Protein Tyrosine Nitration in Mammalian Cells

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Genetically encoded protein tyrosine nitration in mammalian cells Joseph J. Porter, Hyo Sang Jang, Elise M. Van Fossen, Duy P Nguyen, Taylor S. Willi, Richard B. Cooley, and Ryan A. Mehl ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.9b00371 • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 30, 2019

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ACS Chemical Biology

Genetically encoded protein tyrosine nitration in mammalian cells

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Joseph J. Porter, Hyo Sang Jang, Elise M. Van Fossen, Duy P. Nguyen, Taylor S. Willi, Richard

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B. Cooley, Ryan A. Mehl*

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Running title: Genetically encode protein tyrosine nitration in mammalian cells

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*To whom correspondence should be addressed: Dr. Ryan A. Mehl, Department of Biochemistry

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and Biophysics, 2135 ALS, Oregon State University, Corvallis OR 97331-7305 Telephone:

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(541) 737-4429 Fax: (541) 737-0481 Email: [email protected]

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Keywords: 3-nitrotyrosine, 3-nitrophenylalanine, genetic code expansion, oxidative stress, post-

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translational modifications (PTMs), tyrosine modifications

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ACS Paragon Plus Environment

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ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Tyrosine nitration has served as a major biomarker for oxidative stress and is present in

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high abundance in over 50 disease pathologies in humans. While data mounts on specific disease

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pathways from specific sites of tyrosine nitration, the role of these modifications is still largely

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unclear. Strategies for installing site-specific tyrosine nitration in target proteins in eukaryotic

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cells, through routes not dependent on oxidative stress, would provide a powerful method to

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address the consequences of tyrosine nitration. Developed here is a Methanosarcina barkeri

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aminoacyl-tRNA synthetase/tRNA pair that efficiently incorporates nitrotyrosine site-

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specifically into proteins in mammalian cells. We demonstrate the utility of this approach to

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produce nitrated proteins identified in disease conditions by producing site-specific nitroTyr-

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containing manganese superoxide dismutase and 14-3-3 proteins in eukaryotic cells.

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Introduction Tyrosine nitration is an oxidative post-translational modification (Ox-PTM) that has

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served as a biomarker of oxidative stress in a variety of human diseases including

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neurodegeneration, atherosclerosis, and cancer1. While many proteins contain sites of nitration,

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the consequences of Ox-PTM formation at most of these sites remain unexplored. Progress in

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this field has been hampered because Ox-PTMs are installed chemically as opposed to standard

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enzyme directed PTM installation. Ox-PTMs like nitrotyrosine (nitroTyr) are formed through the

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reaction of cellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) with

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proteins. It is difficult to study the effects of site-specific Ox-PTMs since no chemical oxidation

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method is specific to a single amino acid type or location2. To address these challenges, genetic

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code expansion (GCE) is a promising technology that permits the programmable installation of

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single or multiple PTMs site-specifically, thus enabling direct interrogation of the consequences

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of site-specific Ox-PTMs.

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Many reports have proposed the functional importance of tyrosine nitration on proteins

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involved in cell signaling, metabolism, and cellular structure but no direct method is available to

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evaluate which sites of tyrosine nitration have a cellular effect for eukaryotes3-8. Nitration of the

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mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) is known to

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compromise its function and has been implicated in several chronic inflammatory diseases

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including chronic organ rejection, arthritis, and tumorigenesis9. Nitration is also found at key

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regulatory binding sites on all 7 isoforms of the 14-3-3 family of phospho-binding proteins

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which regulate most major cellular functions 10-13. The standard approach to identify these

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modifications is to immunoblot cellular protein for nitroTyr modifications, then the site of

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tyrosine modification is identified via mass spectrometry. It is common to mutate the site(s) of

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tyrosine nitration to phenylalanine and the cells are exposed to the same conditions that caused

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the original modification. The comparison of activity between the tyrosine- and the

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phenylalanine-containing protein can show that removing a site of tyrosine nitration ablates the

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cell phenotype originally seen. This approach is not always feasible for proteins like MnSOD

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and 14-3-3 as conversion of key active-site tyrosine(s) to phenylalanine(s) adversely affect

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protein function. A method to site-specifically install nitroTyr in eukaryotic cells is necessary in

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order to directly demonstrate the impact of a particular site of nitration on protein and cellular

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function.


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GCE uses orthogonal aminoacyl-tRNA synthetase/tRNACUA (aaRS/tRNA) pairs

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engineered for specific non-canonical amino acids (ncAAs)14. This method allows for the

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synthesis of recombinant proteins containing PTMs, providing insights into how these

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modifications regulate protein structure and function15. A GCE system developed for the

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incorporation of nitroTyr has been used to show the functional consequences of nitroTyr

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modification on proteins in vitro16, 17. This tyrosyl-RS/tRNA pair derived from the methanogenic

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archaeon Methanocaldococcus janaschii (Mj) is orthogonal in E. coli and has been used to

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produce a number of site-specifically nitrated proteins for functional studies16, 18, 19. Studying the

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effect of nitration in heat shock protein 90 (Hsp90) in vivo required the expression and

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purification of site-specifically modified Hsp90 from E. coli followed by delivery to eukaryotic

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cells via protein transfection reagent18. While transfection of cells with nitrated protein was

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successful in evaluating the toxicity of site-specifically nitrated Hsp90, studies on cell

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development, protein processing, or transmembrane proteins will require eukaryotic cell

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expression. The Mj nitroTyr-RS/tRNA pair is not orthogonal in eukaryotic cells, necessitating a

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new system for evaluating the effects of nitroTyr in eukaryotic systems. The pyrrolysine

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RS/tRNA pair from several species of methanogenic archaea has emerged as a particularly useful

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platform for GCE as it allows for the evolution of new aaRSs in E. coli and application of the

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evolved aaRS/tRNA in bacterial and eukaryotic cells20.

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Here we develop pyrrolysine aaRS/tRNA pairs that can encode nitroTyr, and the

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structural analogue 3-nitroPhenylalanine (3-nitroPhe). We characterize the efficiency and fidelity

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of these aaRS/tRNA pairs using ncAA-sfGFP expressed in E. coli and mammalian cells. We

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show that functionally efficient pyrrolysine aaRS/tRNA pairs in E. coli do not always function in

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mammalian cells but this limitation can be overcome when the efficiency of the pyrrolysine

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aaRS/tRNA pairs is improved to function with lower concentrations of ncAA. We demonstrate

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the utility of the optimized nitroTyr-RS/tRNA pairs by producing the physiologically nitrated

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proteins, MnSOD and 14-3-3, in mammalian cells and verify the site-specific nitration in vivo.

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The technology to site-specifically install nitroTyr and structural analogues directly into proteins

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in mammalian cells will facilitate an understanding of the structural and functional consequences

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of this Ox-PTM.


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Results and Discussion Selection of an aminoacyl-tRNA synthetase specific for nitroTyrosine and 3-nitroPhenylalanine. To enable studies of nitroTyr modifications on eukaryotic cellular function, we sought to

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identify a Methanosarcina barkeri pyrrolysyl-aaRS/tRNA pair capable of site-specifically

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incorporating nitroTyr into proteins in response to an amber stop codon (TAG). To do this, we

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screened a library of MbPylRS variants in which five active-site residues were randomized to all

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20 amino acids (L270, Y271, L274, N311, C313)21. After a single round of positive selection in

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the presence of nitroTyr or 3-nitroPhe and a single round of negative selection against canonical

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amino acids, 48 colonies were assessed for their efficiency in suppressing a TAG codon

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interrupted sfGFP gene at amino acid site 150 (sfGFP-150TAG) in the presence of nitroTyr and

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3-nitroPhe. Simultaneously, the ability of the selected synthetases to discriminate against

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canonical amino acids was assessed by expressing the sfGFP-150TAG in the absence of ncAA.

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The top 16 performing clones, as based on their efficiency (full-length protein yield) and fidelity

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(level of canonical amino acid misincorporation), were further evaluated at a larger scale in the

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presence of 1 mM nitroTyr and 1 mM 3-nitroPhe. Sequencing of these 16 clones revealed 14

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unique MbPylRS sequences (Supporting Table 1). Of these clones, the selected mutant MbPylRS

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“F4” efficiently incorporated nitroTyr and showed remarkable permissivity for 3-nitroPhe

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compared to the other mutant MbPylRSs (Supporting Figure 1), and was chosen for use in

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mammalian cells. While the F4 MbPylRS efficiently encoded nitroTyr and 3-nitroPhe in E. coli,

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when we tested its ability to incorporate these ncAAs into sfGFP in HEK293T cells only 3-

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nitroPhe was efficiently incorporated (Figure 1 and Supporting Figure 2). In order to select an

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MbPylRS that would efficiently incorporate nitroTyr in eukaryotic cells, the entire pool of

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library members left after the first round of positive and negative selection were subjected to two

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more rounds each of positive and negative selections. The most efficient mutant MbPylRS from

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these rounds of selection “A7” incorporated nitroTyr in E. coli but was not permissive to 3-

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nitroPhe (Figure 1B). The A7 MbPylRS/tRNA pair possesses comparable efficiency and fidelity

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for nitroTyr incorporation in E. coli as compared to the previously evolved Mj tyrosyl-tRNA

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synthetase/tRNA pair (Mj-RS 5b)17.

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Figure 1. Evaluation of top selection hits by expression of TAG-interrupted sfGFP. (A) Structures of nitroTyr and the 3-nitroPhe incorporated via genetic code expansion in this study. (B) Assessment of fluorescence normalized to optical density at 600 nm for cells expressing the sfGFP150TAG gene along with each of the synthetase variants identified from the selection process. Cultures were expressed in the presence of 1 mM nitroTyr (black), 3-nitroPhe (light gray), in the absence of ncAA (grey). The top MbPylRS/tRNA pairs characterized in this study were compared against the previously developed Mj tyrosyl aminoacyl-tRNA/tRNA pair for nitroTyr (5b MjRS). The WT sfGFP positive control represents non-TAG-interrupted sfGFP expression levels achieved by purely natural translation. (C) Coomassie blue stained SDS-PAGE of sfGFP variants expressed and affinity purified from cultures shown in panel B.

We then evaluated the efficiency, fidelity, and permissivity (range of ncAAs

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incorporated) of the F4 and A7 MbRSs in an E. coli sfGFP-150TAG suppression reporter assay.

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The F4 MbPylRS efficiently suppressed sfGFP-150TAG in the presence of 1 mM nitroTyr and

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3-nitroPhe-, while the A7 MbPylRS only suppressed sfGFP-150TAG in the presence of 1 mM

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nitroTyr (Figure 1B). Modified sfGFP was purified to verify the ncAA incorporation did not

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alter sfGFP fluorescence, alter sfGFP solubility and in vivo fluorescence resulted from full-

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length expressed sfGFP containing a C-terminal 6xhis affinity tag. Approximately 340 mg

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(nitroTyr – F4 MbPylRS), 750 mg (3-nitroPhe- F4 MbPylRS), and 140 mg (nitroTyr – A7

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MbPylRS) of sfGFP containing ncAA at site 150 were purified to homogeneity per liter of media

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(Figure 1C). For comparison, WT-sfGFP yielded 500 mg per liter culture under similar

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conditions. No substantial amber suppression was observed in cultures not supplemented with

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ncAA and in vivo sfGFP-fluorescence correlated with purified protein yields. (Figure 1C).

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To confirm nitroTyr and 3-nitroPhe were accurately incorporated into recombinant

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proteins by the F4 and A7 MbPylRSs, we measured the masses of WT-sfGFP, sfGFP-nitroTyr-

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150, and sfGFP-3-nitroPhe-150 using ESI-Q-Tof mass analysis (Supporting Figure 3). Each of


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these variants had the expected masses associated with incorporation of their respective ncAA.

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No mis-incorporation of natural amino acids was detected.

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While E. coli media is generally supplemented with 1 mM ncAA, we wanted to assess

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the concentration of ncAA required for efficient production of ncAA-containing sfGFP using the

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F4 and A7 MbPylRSs for recombinant protein expression in cells. In order to assess this, we

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titrated the amount of nitroTyr or 3-nitroPhe added to the media and measured the amount of

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sfGFP-TAG-150 protein produced. The curves shown in Figure 2 are fit with parameters we

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have termed UP50 (ncAA concentration at which half-maximal sfGFP-150-ncAA is produced)

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and UPmax (maximum amount produced or the yield of sfGFP). These analyses revealed F4

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MbPylRS UP50’s of 90 μM and 600 μM respectively for nitroTyr and 3-nitroPhe. The A7

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MbPylRS UP50 was 3 μM for nitroTyr, while 3-nitroPhe is a very poor substrate for this

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MbPylRS (Figure 2B). Based on the lower UP50 for nitroTyr, we expect that the A7 MbPylRS

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will have a higher yield of nitroTyr-containing protein at lower nitroTyr concentrations in cells.

B

5000 4000 3000 2000

F4 MbRS A7 MbRS

1000 0 0

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OD Adjusted Fluorescence (A.U.)

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OD Adjusted Fluorescence (A.U.)

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0.2

0.4 0.6 [nitroTyr] mM

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1

1.2

16000 12000 8000

F4 MbRS A7 MbRS

4000 0 0

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1 1.5 2 [3-nitroPhe] mM

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In-cell fluorescence was measured to determine relative amounts of sfGFP-150-nitroTyr or sfGFP-150-3nitroPhe protein production in the presence of (A) nitroTyr, or (B) 3-nitroPhe. The curves indicated display the best fit from which the indicated UP50 values were derived.

Figure 2. UP50 determination for nitroTyr or 3-nitroPhe incorporated by the F4 or A7 MbPylRS.

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We have found that nitroTyr sensitive antibodies used to monitor protein nitration show a

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wide range of sensitivities dependent on the nitrated protein as well as the site of nitration. To

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determine the best antibody to monitor site-specifically nitrated proteins in HEK cells we

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prepared nitroTyr- and 3-nitroPhe-containing MnSOD and 14-3-3 proteins in E. coli. All of the

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three antibodies tested were immunoreactive to MnSOD with nitroTyr incorporated at residue 34

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while they displayed minimal immunoreactivity to 3-nitro-Phe installed at the same site


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(Supporting Figure 4A-C). For site-specifically modified human 14-3-3 β protein, only the

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polyclonal nitroTyr antibodies were immunoreactive to 14-3-3 with nitroTyr at site 130, while

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the monoclonal nitroTyr antibody 1A6 was not immunoreactive under the conditions tested

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(Supporting Figure 4A-C). Based on the broad specificity and robust sensitivity we used the

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Millipore polyclonal nitroTyr antibody in future studies to monitor nitroTyr incorporation into

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proteins in eukaryotic cells. The substrates tested demonstrated marked differences in nitroTyr

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antibody sensitivity indicating that those employing nitroTyr antibodies should be mindful to

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determine the sensitivity of the various nitroTyr antibodies to their protein of interest. Finally,

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the Cayman polyclonal nitroTyr antibody displayed a low level of immunoreactivity to human

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14-3-3 β with 3-nitroPhe incorporated at site 130, indicating that the nitrophenol moiety is

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generally necessary for efficient recognitions by the nitroTyr antibodies (Supporting Figure 4C).

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Expression of site-specifically incorporated nitroTyr proteins in eukaryotic cells The MbPylRS/tRNA pair possesses the added utility of usage in eukaryotic cells14, 22

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potentially allowing the study of nitroTyr in its native cellular context. To that end we set out to

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apply this system to the first genetically encoded Ox-PTM in eukaryotic cells. Before

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incorporating nitroTyr in eukaryotic cells, we first determined the maximum allowable

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concentration of nitroTyr for HEK293T cell viability. We found that nitroTyr did not display

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significant toxicity at concentrations up to 0.3 mM at 48 hours after treatment, but cell viability

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was compromised when media was supplemented with 1 mM nitroTyr (Supporting Figure 5).

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Interestingly, 3-nitroPhe showed no apparent effect on the cell density when evaluated up to 1

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mM, indicating that the 3-nitroPhe is notably less toxic than nitroTyr. Based on the HEK293T

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cells toxicity profile for nitroTyr and 3-nitroPhe, future experiments were conducted using 0.3

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mM ncAA in the media.

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We hypothesized that we could take advantage of the highly efficient F4-MbPylRS/tRNA

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pair selected for incorporation of nitroTyr in E. coli and use it for incorporation of nitroTyr in

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mammalian cells. To test this, we cloned a human codon optimized version of the F4 synthetase

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and tRNA into a pAcBac1 mammalian expression vector and in a separate pAcBac1 vector we

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cloned a sfGFP-TAG150 (Supporting Figure 6). To our surprise, the F4-MbPylRS/tRNA pair

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could efficiently incorporate 3-nitroPhe but not nitroTyr in HEK293T cells, as mentioned above

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(Supporting Figure 2). Since the F4-MbPylRS/tRNA pair functioned efficiently with 0.1-1.0 mM

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3-nitroPhe in the media, the GCE machinery is being functionally produced in eukaryotic cells.


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Comparing efficiencies of 3-nitroPhe vs nitroTyr UP50 data, the maximum efficiency of the F4-

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MbPylRS/tRNA pair in E. coli for 3-nitroPhe was three times that compared to nitroTyr (Figure

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2). If there is an ncAA uptake difference between E. coli and HEK cells, this efficiency

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difference at low concentrations of ncAA could be exacerbated. We reasoned that a synthetase

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able to more efficiently incorporate nitroTyr at lower nitroTyr concentrations was needed for

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mammalian cell expression. After selecting and characterizing additional Mb synthetases (see

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Materials and Methods), the A7-MbPylRS was found to be more efficient at lower nitroTyr

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concentrations than the F4-MbPylRS (Figure 2A), and so the gene for it was codon optimized for

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mammalian expression and incorporated into the pAcBac1 plasmid. Indeed, the A7-MbPylRS

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did support robust incorporation of nitroTyr in mammalian cells in the presence of 0.3 mM

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nitroTyr, but not in the absence of ncAA, as shown by fluorescence microscopy and fluorescence

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of crude cell lysate (Figure 3 B-F).


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Figure 3. Expression and optimization of nitroTyr-containing protein in HEK293T cells. (A) Two plasmids were co-transfected for expression of nitroTyr-containing protein in HEK293T cells. The first plasmid (P1) contains two copies of MmPylTc downstream of U6 promoter (U6-Pyl tRNA1), two copies of DhPyl3M downstream of H1 RNA polymerase III promoter (H1-Pyl tRNA2), and sfGFP150TAG downstream of the CMV promoter (CMV). pA indicates bovine growth hormone gene polyadenylation signal. The second plasmid (P2) two copies of MmPylTc downstream of U6 promoter (U6-Pyl tRNA1), two copies of DhPyl3M downstream of H1 RNA polymerase III promoter (H1-Pyl tRNA2), and A7-MbPylRS, downstream of the CMV promoter. HEK293T cells were transfected for 48 h with P1 and P2 using Lipofectamine 2000 (Thermofisher) in the presence (C and E) or absence (B and D) of 0.3 mM nitroTyrosine. Fluorescence images (B and C) and phase contrast images (D and E) were


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captured using EVOS FL cell imaging system (Thermofisher). Scale bar, 50 μm. (F) Cell lysates were prepared in modified RIPA buffer and sfGFP fluorescence was measured using 485 nm excitation and 528 emission. (G) Assessment of production of nitroTyr-containing sfGFP based on the ratio of plasmid containing tRNA and sfGFP-150TAG (P1) to plasmid containing tRNA and A7-MbPylRS (P2). Increasing the ratio of tRNA and sfGFP-150TAG to A7-MbPylRS increases nitroTyr-containing sfGFP production shown here by flow cytometry. The MFI for (+)nitroTyr P2 population was calculated using CytExpert software. One-way ANOVA with Bonferroni post-test. mean±SEM. #, not significant; *, P

Genetically Encoded Protein Tyrosine Nitration in Mammalian Cells

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