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J. Biol. Chem., Vol. 275, Issue 29, 21813-21816, July 21, 2000

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ACCELERATED PUBLICATION
Translational Incorporation of S-Nitrosohomocysteine into Protein^*

Hieronim Jakubowski

From the Department of Microbiology and Molecular Genetics, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103

Received for publication, April 25, 2000, and in revised form, May 11, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The non-protein amino acid homocysteine (Hcy), owing to its structural similarity to the protein amino acids methionine, isoleucine, and leucine, enters first steps of protein synthesis and is activated by methionyl-, isoleucyl-, and leucyl-tRNA synthetases in vivo. However, translational incorporation of Hcy into protein is prevented by editing mechanisms of these synthetases, which convert misactivated Hcy into thiolactone. The lack of efficient interactions of the side chain of Hcy with the specificity subsite of the synthetic/editing active site is a prerequisite for editing of Hcy. Thus, if the side chain thiol of Hcy were reversibly modified with a small molecule that would enhance its binding to the specificity subsite and prevent editing, such modified Hcy is predicted to be transferred to tRNA and incorporated translationally into protein. Here I show that S-nitroso-Hcy is in fact transferred to tRNA by methionyl-tRNA synthetase and incorporated into protein by the bacterium Escherichia coli. S-Nitroso-Hcy-tRNA also supports translation of mRNAs in a rabbit reticulocyte system. Removal of the nitroso group yields Hcy-tRNA and protein containing Hcy in peptide bonds. S-Nitrosylation-mediated translational incorporation of Hcy into protein may occur under natural conditions in cells and contribute to Hcy-induced pathogenesis in atherosclerosis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Aminoacyl-tRNA synthetases (AARSs)¹ establish the genetic code relationships in translation by matching amino acids with their cognate tRNAs (1). The non-protein amino acid homocysteine (Hcy), an obligatory precursor of methionine in all cell types, presents a major problem for translation, because it is misactivated by MetRS, IleRS, LeuRS, ValRS, and LysRS (2, 3). However, the misactivated Hcy is never transferred to tRNA (4, 5). Essentially absolute selectivity of these enzymes against Hcy is maintained by proofreading, or editing, mechanisms that convert the Hcy~AMP intermediate to Hcy thiolactone (6) (Fig. 1). Continuous editing of Hcy is maintained during the process of charging a tRNA with its cognate amino acid by MetRS, IleRS, and LeuRS in vivo (7-12).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Editing of homocysteine by MetRS. After formation of Hcy~AMP in the synthetic/editing active site of MetRS, the side chain of Hcy moves from the specificity subsite to the thiol subsite, which facilitates the formation of Hcy thiolactone (3, 13, 14).

MetRS directs methionine and Hcy into the synthetic and editing pathways, respectively, by employing the following three subsites of the synthetic/editing active site (3, 13, 14): (i) a subsite containing Asp-52 and Arg-233 that bind -amino and -carboxyl groups, respectively, of the amino acid substrates; (ii) the specificity subsite, containing Trp-305, Phe-197, and Tyr-15, that preferentially binds the side chain of the cognate substrate methionine; (iii) the thiol-binding subsite that interacts with the side chain thiol of Hcy and facilitates thioester bond formation during editing; this site also accepts any thiol mimicking the side chain of Hcy. IleRS employs a similar thiol-binding subsite to facilitate editing of Hcy (15).

In the synthetic pathway, intermolecular reaction of the activated carboxyl group of methionine with the 2'-hydroxyl of the terminal adenosine of tRNA^Met yields Met-tRNA^Met. In the editing pathway, intramolecular reaction of the activated carboxyl group of Hcy with the sulfur of its side chain yields thiolactone. Whether an amino acid is transferred to tRNA or edited is determined by the competition for its activated carboxyl group between the side chain of the amino acid and the 3'-terminal adenosine of tRNA (13, 14). MetRS transfers methionine to tRNA, because the side chain of methionine is firmly bound in the specificity subsite of the active site by hydrophobic and hydrogen bonding interactions with Trp-305, Phe-197, and Tyr-15, respectively. In contrast, the side chain of Hcy, missing the methyl group of methionine, cannot interact with the specificity subsite as strongly as the side chain of methionine does. This allows the side chain of Hcy to bind in the editing (or thiol-binding) subsite (3, 14), which facilitates the intramolecular reaction between the activated carboxyl group and the side chain thiol of Hcy (Fig. 1). Thus, keeping the side chain of Hcy in the specificity subsite is predicted to prevent its editing and favor the transfer to tRNA. A possible way to achieve this is by utilizing S-nitrosothiol chemistry. Here I show that MetRS uses S-nitroso-Hcy as a substrate in the tRNA aminoacylation reaction, forming S-nitroso-Hcy-tRNA, and that S-nitroso-Hcy-tRNA transfers S-nitroso-Hcy into protein.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Plasmids and Strains-- Plasmid-encoded gene for Escherichia coli MetRS was overexpressed in E. coli strain JM101 as described before (16). E. coli strain CAG1849/pREP4/pQE15 (metE::Tn10/pREP4/pQE15) (17), which produces mouse DHFR protein upon induction with IPTG was kindly provided by K. Kiick and D. Tirrell (Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA). The strain requires methionine for growth because of the insertion of the transposon Tn10 into the metE gene, which is essential for the final step of methionine biosynthesis.

Methionyl-tRNA Synthetase-- E. coli MetRS was purified to homogeneity from the overproducing strain as described before (13, 14, 16).

Preparation of L-[³⁵S]Hcy Thiolactone-- The method of Baernstein (18), based on the digestion of methionine to Hcy thiolactone in the presence of hydriodic acid, was used with the following modifications. 250 nmol of L-Met was combined with 5 mCi of carrier-free L-[³⁵S]Met (0.3-ml aqueous solution containing 0.1% 2-mercaptoethanol, Amersham Pharmacia Biotech) in a 1-ml glass ampoule and lyophilized on a Speed-Vac. The residue was dissolved in 0.15 ml of 57% hydriodic acid containing 1% hypophosphorous acid (Aldrich). To serve as a refluxing tube, a 12-cm glass tube was attached, by flame-sealing, at the tip of the ampoule. The ampoule was then placed on a heating block, and the digestion was allowed to proceed for 4.5 h at 128 °C. After digestion, the resulting crude preparation of L-[³⁵S]Hcy thiolactone was lyophilized, dissolved in 20 µl of water, and purified by 2D TLC on cellulose plates (20 × 10 cm) (Eastman Kodak Co.) using butanol/acetic acid/water (4:1:1, v/v) as the first-dimension solvent and 2-propanol/ethyl acetate/water (5:5:1, v/v) as the second-dimension solvent (7, 8). Thiolactone spot, localized under UV light and by autoradiography, was scraped off the plate and eluted with 2 mM HCl. The overall yield of the procedure was 65%. The preparation of L-[³⁵S]Hcy thiolactone was at least 96% pure on analytical 2D TLC. Maximum levels of contamination with Met, Hcy, and homocysteine were <1, <0.8, and <1%, respectively.

Preparation of L-[³⁵S]Hcy-- L-[³⁵S]Hcy thiolactone (5 mM, 20,000 Ci/mol) was hydrolyzed to completion by treatment with 0.1 M NaOH at room temperature for 10 min. Because the hydrolysis was carried out in normal room atmosphere, [³⁵S]Hcy was mostly oxidized to [³⁵S]homocystine. The solution was neutralized with 0.1 M NaH₂PO₄, and [³⁵S]homocystine was reduced to [³⁵S]Hcy by treatment with 5 mM dithiothreitol at 37 °C for 15 min. Fresh [³⁵S]Hcy was prepared for each experiment.

Preparation of S-Nitroso-Hcy-- S-Nitrosylation of 1 mM L-[³⁵S]Hcy (20,000 Ci/mol) or 50 mM L-Hcy was carried out using equimolar amounts of NaNO₂ in 0.1 M HCl (19). The solution of L-[S-nitroso-³⁵S]Hcy, neutralized with Na₂HPO₄, can be stored at 70 °C for at least two weeks.

ATP-PP_i Exchange and tRNA Aminoacylation Assays-- All assays were performed at 37 °C in a standard buffer containing 0.1 M potassium-HEPES, pH 7.4, 10 mM MgCl₂, and 0.1 mM EDTA. Reactions were initiated by the addition of MetRS, and four time points were taken at time intervals from 1-8 min. ATP-PP_i exchange assays (6, 13) were performed in the presence of 1 mM [-³²P]ATP (100 Ci/mol, Amersham Pharmacia Biotech), 1 mM PP_i, 0.03-1 mM S-nitroso-Hcy, and 2 µM MetRS. Radiolabeled ATP and PPi, separated by TLC on polyethyleneimine-cellulose plates (Merck) using 3 M ammonium sulfate as a solvent, were quantitated by scintillation counting. Unless otherwise stated, tRNA aminoacylation assays (6, 13) were performed in the presence of 2 mM ATP, 20 µM tRNA^Met or tRNA^fMet (1600 pmol/A₂₆₀, Subriden RNA), 2-160 µM of L-[S-nitroso-³⁵S]Hcy, and 0.5 µM MetRS. For tRNA aminoacylation with methionine, 5 µM [³⁵S]Met and 0.02-2 µM MetRS were used. Radiolabeled aminoacyl-tRNA were precipitated with 5% trichloroacetic acid and quantitated by scintillation counting.

Editing Activity Assay-- Unless stated otherwise, editing activity was measured as amino acid-dependent AMP formation from 1 mM [-³²P]ATP (50 Ci/mol) in the presence of 1 mM amino acid and 0.2 µM MetRS in the standard buffer (16).

Preparation of L-[S-Nitroso-³⁵S]Hcy-tRNA and [³⁵S]Met-tRNA-- Reaction mixtures contained 20 µM tRNA^Met or tRNA^fMet (1600 pmol/A₂₆₀, Subriden RNA), 30 µM radiolabeled amino acid (20,000 Ci/mol), 2 mM ATP, and 0.5 µM MetRS in the standard buffer. After 15 min at 37 °C, reaction mixtures were extracted with phenol (saturated with 0.1 M sodium acetate, pH 5), and aminoacyl-tRNA was recovered from aqueous layers by precipitation with ethanol. Residual low molecular weight components from aminoacylation mixtures were removed by repeated washes with 70% ethanol. The amounts of aminoacyl-tRNA purified by phenol extraction corresponded to the amounts present in the aminoacylation mixtures, as determined by trichloroacetic acid precipitation.

Deacylation of [³⁵S]Aminoacyl-tRNA-- The disappearance of [³⁵S]aminoacyl-tRNA, incubated at 37 °C in the presence or absence of MetRS in the standard buffer, was monitored by trichloroacetic acid precipitation (14). First order rate constants, k, were calculated from reaction half-lives, t_0.5, according to k=ln2/t_0.5. Aliquots of reaction mixtures were also analyzed by TLC (14).

Labeling of E. coli with [³⁵S]Amino Acids-- E. coli strain CAG1849/pREP4/pQE15 was grown at 37 °C in M9 medium supplemented with 0.1 mM methionine, 0.1 mg/ml ampicillin, and 0.035 mg/ml kanamycin (17). After the culture reached cell density of 2 × 10⁸ cells/ml, cells were washed twice with unsupplemented M9 and resuspended in M9 containing 10 µM [³⁵S]amino acid (20,000 Ci/mol) in the absence and presence of 0.5 mM IPTG. To monitor L-[S-nitroso-³⁵S]Hcy and other [³⁵S]metabolites, aliquots of bacterial cultures were subjected to TLC (5, 7, 14).

In Vitro Translation-- Translation of luciferase and globin mRNAs (Promega) was carried out in methionine-free rabbit reticulocyte system according to the manufacturer's procedure (20) using 5 µM L-[S-nitroso-³⁵S]Hcy-tRNA or [³⁵S]Met-tRNA.

SDS-PAGE Analyses-- Products of in vivo and in vitro translation were analyzed on 13% denaturing polyacrylamide gels (21).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

S-Nitroso-Hcy Is a Substrate for MetRS in the Adenylate Formation and tRNA Aminoacylation Reactions-- S-Nitroso-Hcy was a substrate for MetRS in the aminoacyl adenylate formation reaction (Table I). Catalytic efficiency for S-nitroso-Hcy was intermediate between those for methionine and Hcy. The k_cat value for S-nitroso-Hcy (11.2 s¹) was 7- and 2-fold lower than the k_cat values for methionine and Hcy, respectively. The K_m value for S-nitroso-Hcy (0.37 mM) was 18.5-fold higher and 14.3-fold lower than the K_m values for methionine and Hcy, respectively. The enzyme-bound S-nitroso-Hcy~AMP intermediate was relatively stable and, in contrast to the Hcy~AMP intermediate (6), was not edited, as determined by the lack of S-nitroso-Hcy-dependent ATP hydrolysis (Fig. 2B).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   S-Nitroso-Hcy is a substrate for aminoacylation of tRNAMet, but not for editing, catalyzed by MetRS. A, incorporation of [S-nitroso-35S]Hcy (), [35S]Hcy (), and [35S]Met () (30 µM, 20,000 Ci/mol) into tRNAMet catalyzed by 2 µM MetRS. B, S-nitroso-Hcy- () and Hcy-dependent () editing activity was assayed by following the hydrolysis of [-32P]ATP (2 mM, 50 Ci/mol) to [32P]AMP in the presence of 1 mM amino acid and 0.2 µM MetRS.

S-Nitroso-Hcy was also a substrate in the tRNA aminoacylation reaction catalyzed by MetRS (Fig. 2A). Non-saturating kinetics were observed with 2-160 µM S-nitroso-Hcy (not shown). Catalytic efficiencies (k_cat/K_m) indicate that tRNA aminoacylation with S-nitroso-Hcy is about 80-fold less efficient than the tRNA aminoacylation with methionine (Table II). More than 50% of tRNA^Met or tRNA^fMet could be charged with S-nitroso-Hcy (Fig. 2A). As expected (4, 5), Hcy was not incorporated into tRNA (Fig. 2A). Active site mutations in MetRS, known to have detrimental effects on tRNA aminoacylation with methionine (13, 14), had similar affects on tRNA aminoacylation with S-nitroso-Hcy (Table II), suggesting that S-nitroso-Hcy and methionine bind to the same active site (Fig. 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Aminoacylation of tRNA with S-nitroso-Hcy catalyzed by MetRS. After formation of S-nitroso-Hcy~AMP, the side chain of S-nitroso-Hcy does not move to the thiol subsite but remains in the specificity subsite of the synthetic/editing active site of MetRS. This allows the transfer of S-nitroso-Hcy from the adenylate to tRNA.

Properties of S-Nitroso-Hcy-tRNA-- S-Nitroso-Hcy-tRNA, like Met-tRNA, can be isolated from reaction mixtures by phenol extraction. Like Met-tRNA, purified S-nitroso-Hcy-tRNA underwent relatively slow deacylation, both in the absence and presence of MetRS (Table III). In the presence of free thiols, S-nitrosothiols undergo fast transnitrosylation reactions (22). Similarly, S-nitroso-Hcy-tRNA was converted to Hcy-tRNA by treatment with an excess thiol. Hcy-tRNA was about 100 times less stable than S-nitroso-Hcy-tRNA (Table III). Deacylations of Hcy-tRNA and S-nitroso-Hcy-tRNA yielded Hcy thiolactone and S-nitroso-Hcy, respectively, as products (not shown).

Translational Incorporation of S-Nitroso-Hcy into Protein-- Once an amino acid is attached to tRNA, it is destined to become incorporated translationally into protein (23). Thus, the facile aminoacylation of tRNA with S-nitroso-Hcy catalyzed by MetRS suggested that S-nitroso-Hcy could be incorporated into protein. To test this, E. coli strain CAG1849/pREP4/pQE15 (17), which produces mouse DHFR protein upon induction with IPTG was employed. The strain is a methionine auxotroph, unable to metabolize Hcy to methionine because of the insertion of the transposon Tn10 into the metE gene. As shown in Fig. 4A, E. coli utilized S-nitroso-Hcy for incorporation into proteins and, after induction with IPTG, into recombinant mouse DHFR. S-Nitroso-Hcy-DHFR co-migrated with the Met-DHFR on denaturing polyacrylamide gels (Fig. 4B). When [S-nitroso-³⁵S]Hcy-labeled proteins were subjected to hydrolysis by hydrochloric acid (11), [³⁵S]Hcy was released (not shown).

View larger version (45K): [in this window] [in a new window]   Fig. 4.   SDS-PAGE analysis of [S-nitroso-35S]Hcy incorporation into proteins in E. coli. Cultures of E. coli strain CAG1849 (metE::Tn10/pREP4/pQE15) were grown at 37 °C in M9 medium containing 0.1 mM methionine, 0.1 mg/ml ampicillin, and 0.03 mg/ml kanamycin. Bacterial cells were labeled at 37 °C with [35S]amino acids (10 µM, 20,000 Ci/mol) in M9 medium without methionine and antibiotics in the absence and presence of 0.5 mM IPTG. After labeling, bacterial proteins were subjected to SDS-PAGE on 13% polyacrylamide gels. A, proteins form E. coli cells labeled with [S-nitroso-35S]Hcy for 10 min (lanes 1 and 5), 1 h (lanes 2 and 6), 2 h (lanes 3 and 7), and 4 h (lanes 4 and 8) in the absence (lanes 1-4) and presence (lanes 5-8) of 0.5 mM IPTG. B, in control experiments E. coli cells were labeled with [35S]Hcy (lanes 1 and 2) or [35S]Met (lanes 3 and 4) in the absence (lanes 1 and 3) and presence (lanes 2 and 4) of IPTG for 1 h. In experiments with [S-nitroso-35S]Hcy and [35S]Hcy, 10-µl aliquots of labeled cultures were analyzed. In experiments with [35S]Met, 1-µl aliquots of labeled cultures were analyzed. Position of IPTG-inducible mouse DHFR protein encoded by the plasmid pQE15 is indicated.

TLC analyses confirmed that, like other metE strains (7, 10),² the CAG1849 strain metabolized Hcy to thiolactone. Whereas [S-nitroso-³⁵S]Hcy was stable in M9 medium, it was converted into [³⁵S]homocystine and [³⁵S]thiolactone in cultures of E. coli strain CAG1849; no other [³⁵S]metabolites were observed (not shown). The conversion of S-nitroso-Hcy to homocystine in E. coli was most likely because of the presence of enzymes denitrosylating S-nitrosothiols (24, 25). After reduction to Hcy, homocystine is subsequently converted to thiolactone by MetRS, IleRS, and LeuRS in E. coli (7, 10). Because post-translational reaction of thiolactone with protein lysine residues may also lead to incorporation of Hcy into protein (26), control experiments in which E. coli cultures were incubated with Hcy or thiolactone were carried out. There was no incorporation of Hcy (Fig. 4B) or thiolactone (not shown) into bacterial proteins under these conditions. Taken together, these data indicate that S-nitroso-Hcy is incorporated translationally into proteins synthesized by E. coli.

As shown in Fig. 5, S-nitroso-Hcy-tRNA supported translation of globin mRNA and luciferase mRNA in a methionine-free rabbit reticulocyte translation system. Globin and luciferase proteins labeled with [S-nitroso-³⁵S]Hcy were indistinguishable from the corresponding [³⁵S]Met-labeled proteins on polyacrylamide gels. The data thus indicate that S-nitrosylation provides a mechanism for the translational incorporation of Hcy into protein.

View larger version (94K): [in this window] [in a new window]   Fig. 5.   [S-Nitroso-35S]Hcy-tRNA supports translation of luciferase and globin mRNAs in rabbit reticulocyte system. SDS-PAGE analyses of translation mixtures containing 10 µM [35S]S-nitroso-Hcy-tRNA in a rabbit reticulocyte system (Promega) in the absence (lane 3) and presence of globin mRNA (lane 1) or luciferase (Luc) mRNA (lane 2) are shown. Analyses of control reactions containing 10 µM [35S]Met-tRNA in a rabbit reticulocyte system in the absence (lane 6) and presence of globin mRNA (lane 4) or luciferase mRNA (lane 5) are also shown. Aminoacyl-tRNAs used were 1:1 mixtures of charged initiator and elongator methionine tRNAs.

Conclusions-- In cultured human cells, Hcy can be incorporated into proteins post-translationally in a two-step mechanism (11, 26-28). In the first step, Hcy is converted into thiolactone as a result of an error-editing reaction of MetRS. In the second step, thiolactone acylates side chain amino groups of protein lysine residues. Protein damage caused by N-homocysteinylation may underlie involvement of Hcy in vascular diseases (11, 26-28). Because translational incorporation of S-nitroso-Hcy into protein is also likely to lead to protein damage, the present data illustrate an additional mechanism that may explain Hcy-induced pathology associated with atherosclerosis. Specifically, translational incorporation of S-nitroso-Hcy into protein would explain why atherosclerosis is originating mostly at branch points in arteries (29) that are subject to mechanical stress leading to increased production of nitric oxide (19). S-Nitroso-thiols are present in human blood (30), and S-nitroso-Hcy has been detected in cultures of human endothelial cells (19). It is likely that, in subjects with elevated serum Hcy levels, local concentrations of nitric oxide and S-nitroso-Hcy are higher at arterial branch points than elsewhere. Therefore, at branch points, the incorporation of S-nitroso-Hcy into endothelial cell proteins, and resulting protein damage, would be greater than at other points in arteries. Preliminary data suggest that Hcy is incorporated both translationally and post-translationally into proteins from endothelial cell cultures maintained on Hcy in folate-limited media. One cycle of Edman degradation of proteins modified with Hcy thiolactone releases all incorporated Hcy (26). However, similar Edman degradation of Hcy-labeled proteins from endothelial cell cultures releases only 37% of total incorporated Hcy.³ Thus, both translational (via S-nitroso-Hcy) and post-translational (via Hcy thiolactone) incorporation of Hcy into protein provide plausible mechanisms whereby Hcy can affect physiological function.

	ACKNOWLEDGEMENTS

I thank David Tirrell and Kristi Kiick for providing the E. coli strain CAG1849/pREP4/pQE15.

	FOOTNOTES

^* This research was supported by Grant MCB-972-4929 from the National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 973-972-8733; Fax: 973-972-3644; E-mail: jakubows@umdnj.edu.

Published, JBC Papers in Press, May 26, 2000, DOI 10.1074/jbc.C000280200

² H. Jakubowski, unpublished data.

³ H. Jakubowski, unpublished data.

	ABBREVIATIONS

The abbreviations used are: AARS(s), aminoacyl-tRNA synthetase, e.g. MetRS, methionyl-tRNA synthetase; DHFR, dihydrofolate reductase; Hcy, homocysteine; IPTG, isopropyl-thio--galactoside; PAGE, polyacrylamide gel electrophoresis; TLC, thin layer chromatography; 2D, two-dimensional.

	REFERENCES

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