WO2018064571A1 - Procédés de détection de mutagénèse d'adn - Google Patents

Procédés de détection de mutagénèse d'adn Download PDF

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WO2018064571A1
WO2018064571A1 PCT/US2017/054494 US2017054494W WO2018064571A1 WO 2018064571 A1 WO2018064571 A1 WO 2018064571A1 US 2017054494 W US2017054494 W US 2017054494W WO 2018064571 A1 WO2018064571 A1 WO 2018064571A1
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reporter protein
atel
reporter
arginylation
protein
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Fangliang Zhang
Akhilesh Kumar
Michael D. BIRNBAUM
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University Of Miami
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
    • G01N33/5017—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity for testing neoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • C12Y203/02008—Arginyltransferase (2.3.2.8)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/34—Post-translational modifications [PTMs] in chemical analysis of biological material addition of amino acid(s), e.g. arginylation, (poly-)glutamylation, (poly-)glycylation

Definitions

  • NIH/NIGMS GM107333 awarded by the National Institutes of Health/ National Institute of General Medical Sciences. The government has certain rights in the invention.
  • the disclosure relates generally to materials and methods for detecting DNA mutagenesis.
  • Genomic mutation is the direct cause of cancer, amyotrophic lateral sclerosis (ALS), haemophilia, neurofibromatosis, and many other diseases. It is also the driving force of evolution.
  • the frequency of genomic mutation is affected by many environmental factors including chemicals, radiation, and cellular oxidants. Multiple mechanisms exist to regulate and control the effects of those environmental factors in order to keep the rate of genetic mutation at a low level. Failure of any of those mechanisms may lead to diseases.
  • PTM post-translational modifications
  • arginylation is the post-translational addition of an extra arginine to an existing peptide chain, usually to the N-terminus, thus capable of changing the surface charge as well as the primary sequence of the target [1].
  • Arginylation is mediated by arginyltransferase 1 (Atel) [2, 3], an evolutionarily conserved enzyme found in eukaryotic organisms and some bacteria [4-6] .
  • Tel arginyltransferase 1
  • Nearly a hundred proteins have been found to be arginylated and the list of identified substrates is continuously growing [7, 8], suggesting a widespread effect of this PTM in vivo.
  • Arginylation detection using previous methods is difficult, which hinders progress of the field.
  • Previous methods includes sequencing by Mass Spectrometry or by chemical methods such as Edman degradation, and labeling of the target protein with radioactively arginine.
  • Previous methods rely on expensive instruments with limited access or hazardous materials requiring regulatory supervision.
  • Arginylation can be detected by an antibody, which does not require the hazardous chemicals and expensive machinery required by previous methods.
  • ELISA-based assays wherein an antibody specifically recognizing the arginylated form of a short peptide have significant disadvantages. Such an assay can be used to test arginylation of purified (ex vivo) proteins including the Atel enzyme [53].
  • the disclosure provides a method for, e.g., directly evaluating Atel -mediated arginylation inside the cell or in a cell lysate.
  • Measuring and comparing mutation frequency at the single cell level is an important task for research related to cancer and many other diseases.
  • Previous methods developed to quantitatively examine mutation frequency in single cells have limitations.
  • One method utilizes endogenous metabolic enzymes that are either required to detoxify certain toxic chemicals or to generate an essential metabolite.
  • the principle of this type of assay is that a loss-of-function mutation of those genes renders a cell vulnerable to toxic chemicals or make certain metabolites unavailable. Examples of this type of method include assays based on Hypoxanthine Phosphoribosyltransferase (HPRT) gene.
  • HPRT Hypoxanthine Phosphoribosyltransferase
  • detoxification of toxic chemicals or synthesis of metabolites often can be carried out by alternative pathways.
  • Another type of assay uses a single fluorescent or luminescent protein as reporter, which can be examined with optical instruments such as flow cytometers, microscopes, and plate readers.
  • optical instruments such as flow cytometers, microscopes, and plate readers.
  • the principle of this type of assay is that if a mutation enables (on) or disables (off) the light-rendering property of the reporter protein, it can be detected by an optical instrument. While this method requires no toxic chemicals or nutrient-deprivation, the method still suffers from unreliability for failure to distinguish "on'V'off" signals arising from other non-mutation related events, such as a loss of reporter vectors during transient expression, uneven transfection and/or incorporation rates among different cell types, or changes in transcription and translation for the individual reporter gene.
  • the disclosure provides a method for measuring the activity of global arginylation activity, e.g., inside an intact cell, in cell lysates, or in a reconstituted solution with purified proteins. It also provides a method suitable for accurately detecting DNA mutagenesis frequency with a single cell resolution for ratio that is lower than 1 per million.
  • Arginylation activity is globally down-regulated by the deletion or silencing of the ATE1 gene, leading to reduction in cellular sensitivities to a variety of stressing factors, resulting in bypass of growth-arrest or reduction of cell death under stress.
  • the Atel protein level and the global arginylation activity are increased in cells under stress, and Atel mediates cell death in an arginylation-dependent manner. Atel is needed for suppressing the outcome of DNA mutagenesis during DNA-damaging stress.
  • This disclosure describes an example of a post-translational modification having a global effect on DNA mutagenesis.
  • One such method includes introducing into a cell an expression vector comprising a nucleic acid sequence encoding a reporter protein fused to a substrate for arginyltransferase 1 (Atel), such that a reporter fusion protein comprising the reporter protein and Atel substrate is produced.
  • the method further comprises measuring arginylation of the substrate and the reporter protein. The ratio of arginylated substrate to reporter protein is calculated. The ratio is useful for, e.g., characterizing the levels of arginylation.
  • an increase in arginylation in the cell corresponds to increased cell stress.
  • arginylation is detected using an arginylation-specific antibody.
  • the reporter protein is green fluorescent protein (GFP).
  • the arginylation substrate is fused to a cleavable ubiquitin domain located N-terminal to the arginylation substrate.
  • the ubiquitin domain is cleaved by ubiquitin hydrolase.
  • a method for, e.g., estimating DNA mutation frequency (optionally at single-cell resolution) using a reporter plasmid comprises (a) introducing into a cell a reporter plasmid comprising nucleic acids encoding a promoter, a first reporter protein, an internal ribosome entry site (IRES), and a second reporter protein.
  • the first reporter protein and the second reporter protein are different proteins (i.e., the first and second reporter proteins are not both green fluorescent protein).
  • Either the nucleic acid encoding the first reporter protein comprises a premature stop codon and mutation of the stop codon (e.g., to a sense codon) results in the expression of the full-length first reporter protein
  • the nucleic acid encoding the second reporter protein comprises a premature stop codon and mutation of the stop codon results in the expression of the full-length second reporter protein.
  • the method further comprises measuring the first reporter protein and the second reporter protein, and calculating the ratio of first reporter protein to second reporter protein. The ratio allows characterization of the level of mutagenesis of the stop codon within the first or second reporter protein coding sequence, and is indicative of levels of DNA mutagenesis within the cell.
  • the reporter protein sequence lacking the premature stop codon serves as an internal control for transcription and translation.
  • first or second reporter protein is a fluorescent protein; optionally, both the first reporter protein and the second reporter protein are fluorescent proteins.
  • the reporter proteins are green fluorescent protein (GFP) and red fluorescent protein (RFP).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • the technique has a resolution of single cell by nature. The assay results can be detected using instruments with single-cell resolution such as a flow cytometer or an optical microscope to examine the number of cells with the first and the second reporter protein. As such, the method's detection limit is, in various embodiments, better than 1 per million with a false- positive rate lower than 5%, as illustrated in Figure 8.
  • FIGS 1A-1F Knockout or knock-down of ATEl decreases cellular sensitivity towards stressing conditions.
  • the number of viable cells after H 2 0 2 treatment was measured by Calcein AM, a cellular dye that emits fluorescence only in live cells.
  • the number of viable cells after STS and Cdcl 2 treatments was directly counted with an automated cell counter (TC-20 from Biorad) with the cross-staining of Trypan Blue, which stains dead cell but not live cells.
  • Figures 2A-2F Knockout ofATEl in yeast relieves growth-arrest and suppresses cell death during stress response.
  • D) Viability of WT or atelA yeast after indicated hours of treatments with 150 ⁇ CdCl 2 , measured by the colony-forming unit assay and normalized to cells at time 0 (before the application of stressor). Error bars represents SEM (n 3).
  • FIGS 3A-3B Knockout of ATE1 in MEF results in attenuated apoptosis and growth-arrest during stress response.
  • WT or ATE1-KO MEF were treated with STS or CdCl 2 of different concentrations for 5 hours.
  • Propidium iodide (PI) was used to label necrotic and late apoptotic fractions. The cell population is analyzed by FACS. On the left panel representative FACS charts are shown.
  • FIGS 4A-4F The levels of Atel protein and global arginylation activity are up- regulated during stress.
  • A) A scheme illustrating how DD- 15-GFP is used as the reporter of arginylation activity.
  • the fusion protein containing a stretch of 15 amino acids starting with two aspartic acids (D) derived from the N-terminus of mammalian beta-actin, a known substrate of arginylation [31].
  • This peptide is fused with an N-terminal ubiquitin, which is cleaved co-translationally by endogenous de-ubiquitylation enzymes in eukaryotic system and leaves the aspartic acids as the new N-terminus.
  • the arginylation state of this reporter can be probed with an anti-RDD antibody, which only reacts with the arginylated form of the reporter protein.
  • a C-terminal GFP tag is used to facilitate the detection of steady state level of the reporter protein by immunoblotting with anti-GFP antibody.
  • chromosome locus Chromosome VII
  • endo: Atel-GFP The right panels present immunoblots showing the steady-state levels of "endo: Atel-GFP” in yeast treated with increasing concentrations of different stressors: H 2 0 2 (left) or NaCl (right). Tubulin or PGK were used as loading controls.
  • E) WT MEF were exposed to increased concentrations of H 2 0 2 for 30 hours.
  • the arginylation level of the reporter protein was detected by immunoblotting with anti-RDD antibody.
  • the steady-state level of the reporter protein was probed with anti-GFP. Actin antibody was used as loading control.
  • the graph on the right side shows quantification from 4 independent repeats.
  • FIG. 5A-5E The increase of Atel triggers cell death in yeast in a manner that is dependent on its arginylation activity.
  • A) The scheme in the top panel shows the domain structure of plasmid pGALl: ATE1, in which the coding sequence of recombinant protein is preceded by the inducible GAL1 promoter.
  • the picture in the bottom panel shows the growth of atel I. yeast cells carrying either the empty expression vector or pGALl: ATE1 by a serial dilution growth assay on either plate containing glucose (suppressing) or galactose
  • Atel I. yeast cells carrying either the empty expression vector or pGALl: ATE1 in different time points following the initiation of galactose-induced expression, as measured by the numbers of colony-forming cells per OD unit (CFU) that were normalized to starting data point time 0, for Atel or vector control separately. Error bar represents SEM (n> 3).
  • Anti-RDD was used to indicate the level of arginylated reporter.
  • Anti-GFP was used to show the total amount of reporter protein (DD- 15-GFP) in each sample, as well as the total amount of Atel-GFP (either WT or mutant) present in each sample. These two bands were distinguished by their difference in molecular weight (27kD vs. 92kD).
  • FIGS 6A-6D Mammalian Atel is required for cellular sensitivity to stressors in a manner dependent on its arginylation activity.
  • mAtel.l-mut-GFP left panel showing the procedure of using an in- lysate arginylation assay to measure the activities of either the WT version of mAtel.l-GFP or the mutant with cysteine 23 and 26 to serine replacement (referred as mAtel.l-mut-GFP in this study) expressed in stably transformed ATE1 -KO MEF.
  • the arginylation reporter protein, DD- 15-GFP, as described in Figure 4 was expressed and purified from atel I. yeast.
  • Anti-RDD was used to probe the level of arginylation on the reporter protein.
  • Anti-GFP was used to show the total amount of reporter protein (DD- 15-GFP) added in each sample.
  • mAtel antibody was used to detect the level of mAtel.l-GFP (either WT or mutant) present in each sample. Actin was used as loading control for cell lysates.
  • FIGS 7A-7D Knockout of ATE1 increases cell viability upon UV irradiation.
  • C) Quantification of viable cells at 12 hours after UV treatment. Live cells were quantified with cell viability dye Calcein AM and the numbers were normalized to matching samples not irradiated (0 J/m ). Error bar represent SEM (n 3).
  • FIGS 8A-8F Knockout of ATE1 increases mutagenesis upon UV irradiation.
  • the top panel shows a flow chart describing the procedure followed to create isogenic pairs of WT and atelA yeast and for testing emergence of Met-prototrophic mutant colonies on Met- minus plates starting with the same number of cells for UV irradiation.
  • the bottom left panel has representative images showing the auxotrophic colonies emerged from 20 million yeast (in each spreading) without or with a low dose of UV exposure (50 J/m ).
  • the graph on the bottom right is the quantification of the experiment on the left for all tested doses of UV irradiations.
  • the scheme on the right shows a portion of the coding sequences and corresponding amino acids in the original mCherryFP gene and the mutated mCherryFP-STOP gene, where a TGG codon, coding for tryptophan (W), is converted to a TGA stop codon.
  • D Representative FACS charts showing the distribution of cell populations by their green and red fluorescence, for WT or ATE1 -KO MEF, in untreated condition or treated with low-dose UV irradiations that are not expected to lead to significant cell death (two pulses of 20 J/m irradiations over 48 hours, followed by 24 hour recovery).
  • the windows marked "B” were the gate setting used to quantify and sort red- fluorescence-positive cells.
  • Figure 9 The stress-induced increase of arginylation signal is dependent on the presence of Atel.
  • left side shows a similar procedure of using in-lysate arginylation assay on atel- ⁇ yeast exposed to increased duration of 1M NaCl stress.
  • Right side shows a representative immunoblots probed with anti-RDD and anti-GFP, for the detection of arginylated form and the total level of the reporter protein DD- 15-GFP, respectively.
  • the arrows indicated the expected position of the band of DD- 15-GFP on the blots.
  • FIG. 10 Validation of the reverting mutation by DNA sequencing.
  • ATE1-KO cells stably with transfected mutation reporter mCherry-STOP were treated with UV- irradiation as shown in Fig.8 and the red-fluorescence positive cells were enriched by sorting and then grown for up to two weeks to reach sufficient cell numbers.
  • Genomic DNA were extracted from those red-positive cells, as well as from untreated cells (as a control for sequencing).
  • the DNA was amplified with two rounds of nested-PCR with two sets of primers that are specific for the region containing the mCherryFP sequence.
  • the PCR products with anticipated size were then submitted for Sanger sequencing.
  • Trp residue in this location is essential for the fluorescence of mCherryFP, so that only such a revertant can be detected and collected by FACS.
  • the disclosure provides methods for, e.g., detecting DNA mutagenesis.
  • One such method includes introducing into a cell an expression vector comprising a nucleic acid sequence encoding a reporter protein fused to a substrate for arginyltransferase 1 (Atel), such that a reporter fusion protein comprising the reporter protein and Atel substrate is produced.
  • the method further comprises measuring arginylation of the substrate and the reporter protein. The ratio of arginylated substrate to reporter protein is calculated.
  • the method comprises (a) introducing into a cell a reporter plasmid comprising nucleic acids encoding a promoter, a first reporter protein, an internal ribosome entry site (IRES), and a second reporter protein.
  • IRS internal ribosome entry site
  • the first reporter protein and the second reporter protein are different proteins (i.e., the first and second reporter proteins are not both green fluorescent protein).
  • the method further comprises measuring the first reporter protein and the second reporter protein, and calculating the ratio of first reporter protein to second reporter protein.
  • reporter proteins when two reporter proteins are driven by one promoter and separated by an ribosome internal entry site (IRES), no difference in transcription is expected between the reporter proteins; the reporter proteins are located on the same expression vector (or integrated into the same genomic locus, if stable integration is used) and will be transcribed with the same transcription machinery. While a small difference in translational efficiency may occur between the two reporters, the difference does not result in a false signal because, e.g., one reporter protein serves as the mutation target while the other one is an internal control to validate the existence and transcriptional status of the reporter proteins.
  • IRS ribosome internal entry site
  • the reporter assay described herein can be used to reliably and accurately compare mutation frequencies between different cell types and different organisms regardless of the difference in their transfection efficiency and transcriptional or translational activity, which cannot be reliably accomplished with pre-existing methods.
  • Various embodiments of the method of the disclosure involve detection of arginylation.
  • Protein arginylation is a posttranslational addition of an extra arginine to an existing protein substrate, which is mediated solely by arginyltransferase 1 (Atel).
  • Arginylation changes primary sequence as well as surface charge of a protein and, therefore, potentially changes degradation half-life and functions of the target protein.
  • the disclosure provides a reporter fusion protein comprising an N-terminal sequence that is recognized by Atel as its substrate fused to a C-terminus reporter protein (e.g., GFP or other reporter protein that generates a signal that can be detected, preferably detected in an intact cell).
  • the reporter fusion protein is compatible for in vitro and in vivo applications, which is an improvement compared to the peptide-based assay.
  • the arginylated form of the recombinant fusion protein can be recognized with, e.g., an antibody that specifically recognizes the extra arginine within the substrate, while the total amount of the recombinant fusion protein can be measured by another antibody that specifically recognizes the reporter protein subunit of the fusion protein. The ratio of these two signals is then calculated to reflect the actual arginylation extent of the reporter substrate.
  • the use of two antibodies in various embodiments to confirm the arginylation level of the reporter protein increases the specificities and accuracy of this assay. This method is applicable for, e.g., Western Blot format, which allow the user to specifically detect the arginylation signal of the reporter without confusion with other proteins.
  • the method can be used for applications that require close proximity of two or more antibodies to generate a signal for readout, such as, for example, the Alpha Technology/ AlphaScreen.
  • Atel and Ate 1 -mediated arginylation are up- regulated as part of the general stress response to induce cell growth-arrest and cell death, which results in the prevention of DNA mutagenesis under stress. Furthermore the disclosure clarifies the nature of the involvements of Atel and its arginylation activity in stress response, potentially providing a novel explanation for the in vivo functions of Atel and arginylation in a host of human diseases. Many of these stressing conditions are known to have causative effects for human diseases such as cancer, cardiovascular disease, aging, developmental abnormalities, injury and inflammation.
  • Anti-RDD Anti-RDD
  • Genscript INC. Genscript INC.
  • a custom synthesized peptide with the sequence of RDDIAALVVDC (SEQ ID NO: 1) (from Genscript) was conjugated through the C-terminal cysteine to a carrier protein, keyhole limpet hemocyanin (KLH), to increase the presentation of the N-terminus of the peptide.
  • KLH keyhole limpet hemocyanin
  • the conjugated protein was used as the immunogen for repeated immunization in rabbits.
  • the harvested antisera was cross-absorbed by another synthetic peptide with the sequence of DDIAALVVDC (SEQ ID NO:2) (from Genscript).
  • anti-RDD antisera
  • p.LKO lentiviral vectors containing shRNA targeting GFP, mouse Atel, and human Atel were obtained from the Mission shRNA catalog (Sigma; clone NM_007041.1-520slcl for human; clone NM_013799.2-1507slcl for mouse). These vectors were packaged using VSV.g and ⁇ 8.2 lentivirus packaging vectors by co-transfecting HEK293T cells with the aid of transfection kit Lipofectamin 2000 (Life Technologies). The viral supernatant was collected at 24, 48, and 72 hours, and filtered through a 0.45 ⁇ Syringe filter (Olympus, #25-246).
  • the supernatant was then used to transduce MEF and HFF cells, aided by 10 ⁇ g/mL polybrene. After transduction, 5 ⁇ g/mL puromycin was used to select transduced fibroblast cells for 5-10 days. Once the selected line was stabilized, a Western blot was run on a cell lysate to examine changes in Atel levels.
  • the WT cell lysate was mixed with equal volume of lysate of atelA cells expressing the DD-P15-GFP to start the arginylation reaction.
  • the reaction was carried out for 10 minutes at 37°C.
  • the reaction was stopped by the addition of 1/3 volume of 4x SDS sample buffer and boiling.
  • exponential growth-phase cells of MEF were harvested from culture plate, washed by dPBS, and then weighed on scale.
  • the cell pellets were lysed with 2x volume of a modified reaction buffer (50mM TRIS/HCl, 32mM Na 3 P0 4 , pH 7.4, 5mM MgCl 2 , ImM EDTA, 2.5mM ATP, 0.2mM Arginine, with 0.2% NP-40).
  • a modified reaction buffer 50mM TRIS/HCl, 32mM Na 3 P0 4 , pH 7.4, 5mM MgCl 2 , ImM EDTA, 2.5mM ATP, 0.2mM Arginine, with 0.2% NP-40.
  • As an arginylation reporter recombinant protein DD- 15-GFP was expressed in atelA yeast and then purified by GFP-TRAPS nanobody conjugated to magnetic beads (Bulldog Bio) and was shown to be more than 95% pure by Coomassie-blue staining in SDS- PAGE. This protein was added to the cell lysate as the substrate for arginylation. The reaction is allowed to
  • Methods of using the reporter plasmid as described in this patent application may include the following.
  • MEF WT or ATE1-KO
  • Equal numbers of each type of cells were inoculated on 150mm-diameter cell culture dish for 4-6 hours to allow attachment. The final cell density did not exceed 25% confluency.
  • the culture medium is removed. After UV treatment, the original culture medium was added back to the dish and the cells were allowed to recover for 24 hours in C0 2 incubator at 37°C before another round of UV irradiation. After two rounds of UV irradiations, the cells were allowed to recover for 24 hours, before FACS analysis was performed on viable cells that remained attached to the dish.
  • Atel is coded by a single gene in yeast and mammals [4, 5].
  • S. cerevisiae strain BY4741, unless otherwise indicated
  • no obvious effect on growth in non-stressing conditions in nutrient-rich medium Fig. 1A, B.
  • stress including H 2 0 2 -induced oxidative stress, heavy metals, high salt, or high temperature
  • WT wild-type yeast grew at a significantly lower rate compared to non-stressing conditions, which is an expected outcome of normal stress response (Fig. 1A and IB).
  • ATE1 cellular viabilities by colony- formation unit (CFU) in yeast cultures in the presence of lethal doses of H 2 0 2 were examined.
  • CFU colony- formation unit
  • Atel ⁇ yeast cultures were found to have higher percentages of viable cells compared to the WT (Fig. 2A).
  • TUNEL assay to probe apoptosis, a programmed cell death event, the deletion of ATE1 greatly attenuated H 2 0 2 -induced apoptosis (Fig. 2B), which contradicts the prevailing hypothesis for the anti-apoptotic roles for Atel and arginylation [23, 28, 29].
  • atel A yeast were able to form significantly more colonies than the WT yeast, indicating that a deletion of ATE1 in this condition yielded a higher cell survival rate (Fig.2F). Therefore, the lack of Atel may lead to the bypass of growth-arrest and/or the suppression of cell death from the same stressor, dependent on the intensity of stressor.
  • DD-P15-GFP a reporter substrate termed DD-P15-GFP was designed (Fig. 4A).
  • DD-P15-GFP a reporter substrate termed DD-P15-GFP was designed (Fig. 4A).
  • Fig. 4B a reporter substrate termed DD-P15-GFP was designed.
  • Fig. 4E A similar observation was made in mammalian cells MEF (Fig. 4E).
  • the steady state level of the reporter protein, as probed by anti-GFP was obviously lower in WT yeast than in atelA, likely due to an arginylation-mediated degradation in yeast (Fig. 4B).
  • arginylation was demonstrated by radioactively labeled arginine, which can be incorporated into protein by arginylation or translation. To separate these two effects, translation was inhibited by either inhibitors or the depletion of ribosome [9-14]. However most translation inhibitors are unfortunately leaky, and removal of ribosome can collaterally deplete Atel due to their known affinity [37]. To remove these ambiguities, DD- 15-GFP was used for an "in-lysate" reaction to examine arginylation activity in cell extracts (Fig.4C left panel).
  • Atel protein is also up-regulated in stress response
  • a commercially available yeast strain carrying an "z ' n locus" 3 '-end fusion of GFP with the endogenous ATE1 gene (termed “endo: Atel-GFP") under the control of the endogenous ATE1 promoter, was used.
  • the protein level of "endo: Atel-GFP” was proportionally increased with the dose of each stressors such as H 2 O 2 or high salt, (Fig. 4D).
  • probing of the steady-state level of endogenous Atel in MEF resulted in a dose-dependent increase of total Atel protein in the presence of H 2 O 2 , (Fig. 4F).
  • pGAL ATE1-GFP
  • Fig. 5E A similar effect was observed with the induced-expression of a GFP-fused Atel (pGAL: ATE1-GFP) (Fig. 5E).
  • yeast cells were induced in galactose-containing liquid medium for increasing durations. The cell viability was then measured by a CFU assay in glucose-containing agar plates (where the galactose-induction is terminated). It was observed that that the viability of cells carrying the pGAL: ATE1-GFP was dramatically decreased along the time line of galactose induction, suggesting that the up-regulation of Atel is indeed capable of inducing cell death in yeast (Fig. 5B).
  • the in-lysate arginylation assay was utilized with DD- 15-GFP reporter and it was found that the activity of the mutation is less than 25% of the WT enzyme (Fig. 5D).
  • the C20,23S mutation was found to significantly reverse the repressing effects of Atel over-expression in cells (Fig. 5E). Therefore, the effects of yeast Atel on stress response is largely (if not completely) dependent on its arginylation activity.
  • mAtel.l splicing variant 1 of mouse Atel
  • the C-terminal GFP fused recombinant protein was reintroduced into ATE1 -KO MEF by a stable low-expression system that generates recombinant proteins at a level comparable to the endogenous level of WT Atel (Fig.
  • mAtel. l was able to reinstall cellular sensitivity to stressors, such as STS and CdCl 2 (Fig. 6C and 6D), to levels close to WT cells, indicating that mammalian Atel is indeed mediating cell death in stress response.
  • Mammalian Atel also carries two cysteine residues (C23, C26 in mAtel) corresponding to the C20, C23 residues in yeast Atel. Consistent with our observations in yeast Atel, when these two cysteine residues were changed to serine in mouse Atel, the resulted mutant (mAtel.1-mut) had compromised arginylation activity (Fig.
  • Atel is essential for the suppression of mutagenesis during DNA-damaging stress
  • ATE1 -deletion in yeast or MEF was found to significantly increased their resistance to UV light (254 nm UV-C) compared to WT (Fig. 7A, 7B, and 7C).
  • sensitivity to UV in ATE1-KO MEF can be sufficiently reinstalled by the reintroduction of recombinant Atel (mAtel. l). This installation effect was nearly abolished when the catalytically impaired mutations (C23-26S) was introduced in Atel (Fig. 7D).
  • a mutation reporter Met-STOP was designed, in which a Tryptophan (TRP/W) residue in Metl5 gene is replaced with an interrupting STOP codon.
  • TRP/W Tryptophan residue in Metl5 gene is replaced with an interrupting STOP codon.
  • the product of this engineered gene is not expected to rescue the methionine (Met)-auxotrophy phenotype of
  • atel A BY4741 -yeast carrying this reporter plasmid similarly generated negligible numbers of colonies on culture plates in the absence of methionine.
  • atelA yeast produced significantly more colonies acquiring Met-prototrophic phenotype compared to the WT, suggesting that the deletion of Atel lead to higher mutation frequencies (Fig. 8B). Since significant differences between WT and atel A yeast were observed in both low and high doses of UV irradiation, it is likely that both the effects of Atel in growth-arrest and cell death contribute to the mutation suppression.
  • mCherryFP-STOP-IRES-GFP was created, in which an interrupting STOP codon replaces a Trp residue in mCherryFP gene, which is followed by an internal ribosome entry site (IRES) and a green fluorescent protein eGFP (as the internal control for transcription and expression).
  • IRS internal ribosome entry site
  • eGFP green fluorescent protein
  • the WT or ATEl-KO MEF stably transfected with the reporter were analyzed in fluorescence flow cytometry (FACS).
  • FACS fluorescence flow cytometry
  • both the WT and ATEl-KO cells had negligible numbers of red fluorescent cells.
  • a significantly higher ratio of red fluorescent cells was detected in the ATEl-KO cells compared to the WT cells (Fig. 8D and 8E).
  • the positive cells were sorted and subsequently examined by microscopy.

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Abstract

La présente invention concerne un procédé comprenant l'introduction dans une cellule d'un vecteur d'expression comprenant une séquence d'acide nucléique codant pour une protéine rapporteur fusionnée à un substrat d'arginyltransférase 1; la mesure des taux d'arginylation et de la protéine rapporteur; et le calcul du rapport entre le substrat arginylé et la protéine rapporteur. L'invention concerne également un procédé comprenant l'introduction dans une cellule d'un plasmide rapporteur comprenant un acide nucléique codant pour un promoteur, une première protéine rapporteur, un site d'entrée interne des ribosomes (IRES), et une seconde protéine rapporteur, l'acide nucléique codant pour la première protéine rapporteur comprenant un codon d'arrêt prématuré et la mutation du codon d'arrêt conduisant à l'expression de la première protéine rapporteur, ou l'acide nucléique codant pour la seconde protéine rapporteur comprenant un codon d'arrêt prématuré et la mutation du codon d'arrêt conduisant à l'expression de la seconde protéine rapporteur; la mesure de la première protéine rapporteur et de la seconde protéine rapporteur; et le calcul du rapport entre la première protéine rapporteur et la seconde protéine rapporteur.
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Citations (3)

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US20060084097A1 (en) * 2002-03-21 2006-04-20 California Institute Of Technology Modulation of nitric oxide signaling through signaling through specific regulation by arginylation and the N-end rule pathway
US20060234313A1 (en) * 2002-03-21 2006-10-19 California Institute Of Technology, Modulation of angiogenesis through targeting of arginyl transferase (ATE1)
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KUMAR ET AL.: "Posttranslational arginylation enzyme Ate1 affects DNA mutagenesis by regulating stress response", CELL DEATH DIS., vol. 7, no. 9, 29 September 2016 (2016-09-29), pages e2378, XP055604326 *
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