Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9585–9589, September 1997 Biochemistry

The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo

JACKOB MOSKOVITZ,BARBARA S. BERLETT,J.MICHAEL POSTON, AND EARL R. STADTMAN

Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20982-0320

Contributed by Earl R. Stadtman, July 1, 1997

ABSTRACT A gene homologous to methionine sulfoxide MATERIALS AND METHODS reductase (msrA) was identified as the predicted ORF (cosmid 9379) in chromosome V of Saccharomyces cerevisiae encoding Materials. The compound 2,2-azobis-(2-amidino-propane) a protein of 184 amino acids. The corresponding protein has dihydrochloride (AAPH) was purchased from Wako Chemi- been expressed in Escherichia coli and purified to homogene- cals (Richmond, VA). Hydrogen peroxide was purchased from ity. The recombinant yeast MsrA possessed the same substrate Fisher. Dabsyl-chloride was purchased from Pierce. specificity as the other known MsrA enzymes from mamma- S. cerevisiae Peptide Met(O) Reductase: Cloning and Over- lian and bacterial cells. Interruption of the yeast gene resulted expression. Searching GenBank for a yeast homologue to the in a null mutant, ⌬msrA::URA3 strain, which totally lost its bovine msrA cDNA (GenBank accession no. U37150) revealed cellular MsrA activity and was shown to be more sensitive to an ORF of 552 bp that had 32% identity over its whole length oxidative stress in comparison to its wild-type parent strain. to the cDNA sequence of the bovine msrA (GenBank accession Furthermore, high levels of free and protein-bound methio- no. U18796, cosmid 9379, denoted as a homologue of pilB). nine sulfoxide were detected in extracts of msrA mutant cells This ORF was amplified by PCR using S. cerevisiae DNA as relative to their wild-type parent cells, under various oxidative template, a 5Ј sense primer (H1) containing a BamHI site stresses. These findings show that MsrA is responsible for the (5Ј-CTGGAGGGATCCATGGCTGTCGCTGCCAAC), and reduction of methionine sulfoxide in vivo as well as in vitro in a3Јreverse complement primer (H2) with HindIII site (H2: eukaryotic cells. Also, the results support the proposition that 5Ј-AGGGCAAAGCTTCTAAAAAAGCTACATTTC). MsrA possess an antioxidant function. The ability of MsrA to PCR was performed for one cycle of 5 min at 94°C, followed repair oxidative damage in vivo may be of singular importance by 30 cycles of 30 sec at 94°C, 60 sec at 50°C, and 90 sec at 72°C. if methionine residues serve as antioxidants. Both the amplified product and pQE-30 (Qiagen, Chatsworth, CA) were digested with BamHI and HindIII, and the PCR fragment encompassing the complete yeast msrA coding re- Methionine (Met) oxidation is mediated by various biological gion was ligated into the restricted pQE-30 by using T4 DNA oxidants such as hydrogen peroxide, hydroxyl radicals, ozone, ligase (Boehringer Mannheim). E. coli cells (M15) were trans- peroxynitrite, and hypochlorite, as well as by metal catalyzed formed with an aliquot of the ligation mixture and grown in oxidation systems. Peptide methionine sulfoxide reductase Luria–Bertani medium containing ampicillin (100 ␮g͞ml) and (MsrA) is capable of reducing either free methionine sulfoxide kanamycin (25 ␮g͞ml). When cells reached an A of 0.8, [Met(O)] or protein-bound Met(O) to Met, in vitro (1). 600 isopropyl ␤-D-thiogalactoside (IPTG) was added to a final Recently, the mammalian msrA cDNA has been cloned (1) and concentration of 1 mM, and the growth continued for an its protein has been shown to be highly expressed in renal additional 5 hr. The cells were harvest by centrifugation and medulla, retinal pigmented epithelial cells (RPE), blood, and resuspended in 50 mM Na-phosphate (pH 8.0) and 300 mM alveolar macrophages (2). Both macrophages and RPE cells NaCl (buffer A), and sonicated. The lysate was centrifuged, the have the abilities to produce oxidants (3, 4), and their high level supernatant was applied to Ni-NTA resin (Qiagen), and of MsrA is probably maintained to provide an efficient mech- following extensive washing with buffer A, the protein was anism for restoration of intracellular Met(O) to Met. In eluted with buffer A containing 400 mM imidazole. The purity addition to its role in repairing oxidative Met damage to of the protein was analyzed by SDS PAGE. protein, the MsrA in kidney may perform a salvage function by ͞ Disruption of msr A Gene in Yeast Cells. The disruption of converting the Met(O) to Met and thereby sparing the need for msrA was performed according to the procedure developed by replacement of Met lost by oxidative processes. An Escherichia H. Nelson and N. Nelson (personal communication). The coli msr A null mutant has been shown to be more sensitive to msrA gene was interrupted by insertion of URA3 gene in the oxidative stress caused by hydrogen peroxide than the parent middle of the gene after a small deletion, as follows. The strain (5). It has been suggested that MsrA repairs oxidative marker gene (URA3) was cloned in the HindIII site in pBlue- damage to Met that occurs in vivo. This function of MsrA in script and amplified by PCR using the T3 and T7 primers. One vivo has special importance if Met residues act as endogenous pair of primers, A1 and A2, were made at the 3Ј end of the antioxidants as proposed by Levine et al. (6). N-terminal piece of the msrA gene and 5Ј end of the C-terminal In this study we use Saccharomyces cerevisiae as a model for piece, respectively, each containing a specific part of A1 or A2 oxidative stress in a eukaryotic system. First, the yeast msrA fused with the complement sequence of T3 and T7, respec- gene has been cloned and overexpressed in E. coli and the tively. The latter pair is used with H1 and H2 in the first set of resulting recombinant protein has been characterized. Then, a PCRs to produce the N-terminal and C-terminal pieces of the yeast null msrA mutant has been made and its growth and its msrA gene as follows: (i) PCR containing H1 A1 primers cellular pool of Met(O) have been monitored relative to the ϩ with the msrA gene which resulted in the N-terminal piece; and parent strain under various culture conditions. (ii) PCR containing H2 ϩ A2 primers with the msrA gene

The publication costs of this article were defrayed in part by page charge Abbreviations: AAPH, 2,2-azobis-(2-amidino-propane) dihydrochlo- payment. This article must therefore be hereby marked ‘‘advertisement’’ in ride; Met, methionine; Met(O), methionine sulfoxide; MsrA, peptide accordance with 18 U.S.C. §1734 solely to indicate this fact. methionine sulfoxide reductase enzyme; yeast strain ⌬msrA::URA3, 0027-8424͞97͞949585-5$0.00͞0 yeast null mutant of peptide methionine sulfoxide reductase gene; PNAS is available online at http:͞͞www.pnas.org. IPTG, isopropyl ␤-D-thiogalactoside.

9585 Downloaded by guest on September 25, 2021 9586 Biochemistry: Moskovitz et al. Proc. Natl. Acad. Sci. USA 94 (1997)

which resulted in the C-terminal piece. The second set of PCRs solution of Met(O) in 100 mM bicarbonate buffer (pH 9.0) were as follows: (i) PCR containing the H1 ϩ T7 primers with with dabsyl-chloride in acetonitrile. After 10 min at 70°C, the the URA3 gene and the N-terminal piece which resulted in the dabsyl-Met(O) was purified from the reaction mixture by N-terminal piece fused to the URA3 gene; and (ii) PCR passage through a silica gel column and step-wise elution with containing H2 ϩ T3 primers with the URA3 gene and the aqueous solutions of methanol and methanol-acetonitrile. C-terminal piece which resulted in the C-terminal piece fused Purity of the eluates were checked by thin-layer chromatog- to the URA3 gene. The third PCR consisted of H1 ϩ H2 raphy on silica gel plates using the solvent n-butanol͞acetic primers with the products of the second PCR set which acid͞water (60:12:25). The dabsyl-Met(O) was collected by resulted in the final construct: N terminus msrA ϩ URA3 ϩ evaporating the solvent and stored at room temperature. For C-terminal msrA (MUM). This construct was used for yeast measuring MsrA activity in yeast the cells were disrupted in 25 transformation. mM Tris⅐HCl (pH 7.5) in a French pressure cell. All PCRs were performed as above, and the sequences of the Determination of Met(O) in Yeast Extracts. Yeast cells were T7, T3, A1, and A2 primers are as follows: T3, ATTAAC- grown aerobically in synthetic complete medium at 30°C with CCTCACTAAAG; T7, AATACGACTCACTATAG; A1, or without H2O2 (1 mM) or AAPH (6 mM). When cell density TTCCCTTTAGTGAGGGTTAATGGATACTTGTAAA- reached 300 Klett units (turbidity measured in a Klett– ACCTCCGC; and A2, GCCCTATAGTGAGTCGTATT- Summerson colorimeter at 540 nm) cells were spun down and GAATCCATGATCCTACTAC. washed five times with PBS prior to their disruption in French Yeast Strains and Analysis of Mutants. S. cerevisiae haploid pressure cell in buffer B [6 M guanidine chloride͞500 mM strains H8 (Mata ura3–52 his5) and H9 (Mat␣ ura3–52 his6 K-phosphate (pH 2.5)]. Following centrifugation at 20,000 ϫ leu2) were the original strains used in this study. These strains g for 20 min the supernatant solutions were passed through were a gift from Alan Hinnebusch (National Institutes of microconcentrators (microcon 3; Amicon). In each case, the Health). Both strains were transformed with the final PCR flow-through was collected for free amino acid analysis product (MUM) by the lithium acetate method (7), and were whereas the retained material was kept for protein bound grown on minimal media plates containing 0.67% yeast nitro- amino acid analysis, after extensive washing with buffer B. The gen base, 2% dextrose, 2% agar, and the appropriate nutri- protein moiety of each preparation was subjected to CNBr tional requirements without uracil. Several colonies were cleavage as described to quantitate the oxidized Met (6). CNBr collected and grown on minimal medium without uracil, cleaves peptide bonds on the carboxyl side of Met [but not of genomic DNA was isolated from the different transformants, such bonds involving Met(O)] to yield homoserine (10). Hy- and the presence of disrupted msrA gene was assayed by PCR. drogen chloride hydrolysis and amino acid analysis were As shown in Fig. 2, the expected fragments of Ϸ550 bp (msrA carried out on samples with and without CNBr treatment, as gene) in wild types and 1,800 bp (Ϸ550 bp of the msrA gene described (11). plus Ϸ1,250 bp of the URA3 gene) in the ura-disrupted mutants appeared following agarose gel electrophoresis of the RESULTS PCR products. The msrA activity in the different yeat strains was assayed as described below. Isolation of the Yeast msrA Gene and Expression, Purifi- Determination of MsrA Activity. The ability of Met(O) cation, and Characterization of the Recombinant MsrA Pro- reductase to reduce free Met(O) was assayed by using tein. The yeast msrA gene was cloned by PCR method from [3H]Met(O) as substrate, prepared as described by Brot et al. yeast genomic DNA using the sequence at chromosome 5 (8). The reaction mixture (30 ␮l) contained 15 mM DTT, 25 (GenBank accession no. U18796). An ORF of 184 amino acids mM Tris⅐HCl (pH 7.5), 16.7 ␮Mof[3H]Met(O), and yeast (calculated molecular mass of 21,140 Da) showed high homol- extract or pure MsrA. Following incubation at 37°C the ogy (Ϸ40%) to the pilB sequence that had been shown to be reaction was stopped by adding 0.33 mM of Met(O), and the msrA of Neisseria gonorrhoeae (12). The corresponding conversion of [3H]Met(O) to [3H]Met was analyzed by thin- protein was overexpressed in E. coli and purified to homoge- layer chromatography on a silica gel plate using the solvent neity (Fig. 1). The ability of this protein to reduce protein- n-butanol͞acetic acid͞water (60:12:25). After ninhydrin treat- bound Met(O) and free Met(O) to Met was investigated by ment of the plate, the spot that corresponded to the migration using dabsyl-Met(O) or N-acetyl-Met(O) and free Met(O) as of Met was extracted by water and the radioactivity was substrate, respectively. The recombinant protein was found to measured. The reduction of protein-bound Met(O) by MsrA reduce both classes of substrates as shown in Table 1. These was assayed using either N-acetyl[3H]Met(O) (8) or dabsyl- results confirmed that the yeast homologue to pil B is actually Met(O) (9) as substrate. In the latter assay the amino group of the yeast msrA. the Met(O) had been derivatized with dabsyl chloride (4-N,N- Analysis of MsrA Activity in Yeast msrA Null Mutants and dimethylaminoazobenzene-4Ј-sulfonyl chloride). Reduction of Their Parent Strains. To investigate the effects of oxidative the dabsyl-Met(O) to dabsyl-Met was determined by means of stress on yeast cells lacking the msrA gene, a yeast null mutant an HPLC technique. Reaction mixtures (100 ␮l) containing 20 of msrA was constructed as described. To confirm that these mM Tris⅐HCl (pH 7.5), 10 mM MgCl2, 30 mM KCl, 20 mM yeast strains were indeed msrA null mutants, their extracts DTT, 1 ␮M dabsyl-Met(O), and an aliquot of purified MsrA were assayed for msrA activity. As shown in Table 1, the msrA or yeast extract were incubated at 37°C for 30 min and stopped mutant strains lost their ability to reduce protein-bound by the addition of 200 ␮l acetonitrile. After centrifugation for Met(O) and retained only Ϸ33% of their activity toward free 5 min (top speed in a Beckman Microfuge 12 centrifuge), 10 Met(O) relative to their parent strains (Fig. 2). In addition, ␮l of the clear supernatant solution was injected onto a 10-cm rabbit antibodies raised against the recombinant yeast MsrA C18 column (Apex, Jones Chromatography, Denver, CO) protein showed no immunological cross reactivity with cell equilibrated at 50°C with buffer [0.14 M sodium acetate, 0.5 extracts of ⌬msrA::URA3 null mutants (data not shown). ml͞liter triethylamine (pH 6.1)] containing 30% acetonitrile. Effect of Oxidative Stress on Growth and Met(O) Accumu- Using a linear gradient from 30 to 70% acetonitrile over 11 min lation. Under normal growth conditions, the ⌬msrA::URA3 the dabsyl-Met(O) was eluted at 2.6 min whereas the dabsyl- mutant and its parent strain exhibited similar growth pattern. Met was eluted at 5.0 min, as monitored by peak integration After a lag of about 5 hr both strains grew at identical rates at 436 nm (1 pmol of dabsyl-Met gives 340 area units). The (Fig. 3). However, in the presence of H2O2 growth of the column is washed for 5 min with the solvent containing 70% parental strain was only slightly delayed (1–2 hr), whereas acetonitrile and re-equilibrated at 30% before the next injec- growth of the msrA mutant was delayed for an additional 4–5 tion. The dabsyl-Met(O) was prepared by reacting an aqueous hr. Similar results were obtained with the H8 strain and its Downloaded by guest on September 25, 2021 Biochemistry: Moskovitz et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9587

FIG. 2. Interruption of the msrA gene in yeast cells. Haploid strains H8 and H9 were transformed with a DNA fragment containing the disrupted msrA gene by the URA3 gene insertion. The cells were grown on minimal medium plates without uracil. Several colonies were collected and grown on minimal liquid medium without uracil. DNA was isolated from the different transformants, and the presence of disrupted msrA gene was assayed by PCR. PCR products are shown in the agarose gel, using oligonucleotides H1 and H2, of the wild-type and the disruptant strains and the original DNA construct used for the disruption of the gene. Lanes: 1, DNA fragment used for the inter- ruption of the gene; 2 and 4, PCR product of DNA isolated from H8 FIG. 1. SDS͞PAGE analysis of fractions during the purification of and H9 wild-type cells, respectively; 3 and 5, PCR product of DNA recombinant yeast MsrA protein. M15 cells were transformed with isolated from msrA disruptant cells of H8 and H9 strains, respectively. pQE-30 harboring the yeast msrA gene and the expressed protein was purified as described. Lanes: 1, S-30 Ϫ IPTG; 2, S-30 ϩ IPTG; 3, yeast oxidation of protein-bound Met than is the wild-type parent MsrA protein eluted from the Ni-NTA resin after treatment with strain under the oxidative stress conditions examined. It is also buffer A containing 400 mM imidazole. The arrow indicates where MsrA migrates. evident that hydrogen peroxide is more potent than AAPH in causing Met oxidation in proteins, in vivo. msrA mutant (data not shown). In contrast, when the radical- Oxidation of Free Met. The difference between the wild- generating compound AAPH was added to the growth me- type and the mutant msrA yeast strains, in regard to Met dium, the growth of the msrA mutant was only slightly retarded oxidation, was much more pronounced when the content of relative to the wild-type cells (data not shown). In each case the free Met(O) was measured in these cells under the same cells were harvested when the culture reached a density of 300 conditions as described above. As shown in Fig. 4B, under Klett units. Following extensive washing the cells were resus- normal culture conditions without addition of oxidants, no pended in buffer B and extracts were made by passage through free Met(O) was detected in either the msrA mutant or its a French pressure cell. Amino acid composition of the protein parent yeast strain. In contrast, the highest value of free moiety and the free amino acid content in each extract was Met(O) (nmol͞mg dry weight) was detected in the msrA determined. mutant strain when H2O2 was added to the culture medium. Oxidation of Protein Met Residues. As shown in Fig. 4A, under normal conditions the Met(O) accounted for only 2.5% of the total protein Met [Met(O) ϩ Met]. However, after growth in the presence of H2O2 Met(O) accounts for 7.5% and 15% of the total protein Met residues in the H9 and its msrA null mutant, respectively. The treatment with AAPH resulted in a similar pattern but the level of Met(O) was lower, accounting for only 3% and 5% in the H9 and H9⌬msrA::URA3 cells, respectively. These results show that the msrA null mutant of yeast is about twice as sensitive to

Table 1. Methionine sulfoxide reductase (MsrA) activity Specific activity, pmol͞min͞mg protein

Enzyme Dabsyl-Met(O) L-(3H) Met(O) Recombinant yeast MsrA 43,074 4,256 Extract of yeast parent strain 68 15 Extract of yeast msrA mutant 0 5 FIG. 3. Growth of yeast strains in the presence and absence of The MsrA activity was determined as described. The results for the H2O2. Yeast from stationary phase cultures were inoculated into yeast yeast extracts represent both the H8 and H9 strains and their msrA– synthetic medium at 1:300 dilution and were grown aerobically at 30°C null mutants, respectively. Similar results to the activities shown with with or without H2O2 (1 mM). The symbols are defined as follows: the dabsyl-Met(O) substrate were obtained with N-actyl-Met(O) (data wild-type parent strain (H9) grown with (■) or without (ᮀ)H2O2; and not shown). H9 ⌬msrA::URA3 strain grown with (F) or without (E)H2O2. Downloaded by guest on September 25, 2021 9588 Biochemistry: Moskovitz et al. Proc. Natl. Acad. Sci. USA 94 (1997)

toward N terminus blocked Met(O) while retaining only Ϸ33% of the activity toward free Met(O) in comparison to the extracts of its parent strain. These results are in agreement with previous results obtained with the E. coli msrA null mutant (5). In yeast as in E. coli, there are at least two Met(O) reductases, one (MsrA) is able to reduce both free and protein-bound Met(O) and the other that can reduce only free Met(O). In the upstream region of the msrA ORF (ATG 17177 at chromosome V), three sequences containing the TATA box motif have been identified. One (TATA) starting at Ϫ42, and the other two (CATATATA) at positions Ϫ72 and Ϫ114, respectively. These sequences could be the binding sites for RNA polymerase or other transcription factors. Further study is needed to establish the function of these sites. It is evident from the growth patterns that the yeast msrA mutant is more severely inhibited by H2O2 treatment than the wild-type parent strain (Fig. 3). In contrast, the msrA mutant and wild-type strains of yeast are almost equally susceptible to growth inhibition by AAPH (data not shown). The differential effect of H2O2 and AAPH on growth of mutant and wild-type strains may reflect difference in their specificity for amino acid residue oxidation. H2O2 has little ability to directly oxidize amino acid residue other than Met or Cys residues. However, in addition to Met and Cys, free radicals (ROO⅐,RO⅐) formed in the decomposition of AAPH (13) can also oxidize trypto- phan, tyrosin, phenylalanine, and histidine residues in proteins (Y. Ma and E.R.S., unpublished results). This could explain why growth of the msrA mutant is more susceptible to inhi- bition by H2O2 than is the wild type. MsrA can repair growth-limiting oxidation of Met residues in the presence of H2O2 but cannot repair damage to other amino acids residues that limit growth in the presence of AAPH. Nevertheless, FIG. 4. Met(O) content in yeast strains that were grown under different oxidative stress conditions. Each yeast strain was generally exposure of yeast to either H2O2 or AAPH leads to a sub- grown in a synthetic medium as described in Fig. 3 until the cell density stantial increase in the cellular levels of both free and protein reached 300 Klett units under each culture condition. Then cells were bound Met(O). However, in all cases the increasing levels of harvested and their extracts were measured for protein-bound Met(O) Met(O) in msrA mutants are considerably greater than in the (A) or free Met(O) (B), as described. Filled bars represent wild-type parent wild-type strains (Fig. 4). From these results it is clear (H9) strain and hatched bars represent H9 ⌬msrA::URA3 strain. that free and protein-bound Met(O) are substrates for the Similar results were obtained with the H8 yeast strain and its corre- MsrA enzyme in vivo. sponding null msrA mutant strain. H2O2 and AAPH concentrations The oxidative stress-induced increase in free Met(O) is were 1 mM and 6 mM, respectively. likely due to both an increase in the rate of free Met oxidation Also, the ratio of free Met(O) in the msrA mutant to its parent and the release of Met(O) from oxidized proteins as a con- sequence of accelerated proteolytic degradation. Indeed, ox- wild-type strain was Ϸ7:1 under these conditions (Fig. 4B), idation of Met residue in protein has been shown to increase which was about 3-fold higher than the ratio observed with the their susceptibility to proteolysis by the multicatalytic protease protein Met(O) (Fig. 4A). When AAPH was used to induce (6). In addition, the increase in free Met(O) in the msrA oxidative stress the amount of free Met(O) in the msrA mutant mutant may reflect up-regulation of de novo Met synthesis to was much lower than that obtained with H O , and no free 2 2 compensate for the loss of the ability to reduce Met(O) and Met(O) was detected in the wild-type strain (Fig. 4B). These consequently a decrease in the steady-state level of free Met results clearly show that Met(O) accumulated in proteins as needed for protein synthesis. The latter possibility is consistent well as in the free amino acid pool of the yeast strain lacking with the observation that the level of total free Met [Met(O) the msrA gene. ϩ Met] is 6-fold higher in the msrA mutant than in the wild-type strain when grown in medium containing H2O2 or DISCUSSION AAPH (data not shown). In view of the fact that Met residues are among the first to In this study we describe the cloning and disruption of the yeast be attacked by almost all forms of reactive oxygen species msrA gene. The cloned yeast gene had high homology (Ϸ30– ⅐ ⅐ ⅐ Ϫ (HO ,O3,H2O2, ROOH, ROO ,RO, HOCl, ONOO , etc.), 40% identity) to previously described msrA genes from E. coli and that oxidized Met residues are readily reduced back to Met (5), Bos tauros (1), N. gonorrhoeae (12), and Streptococcus by MsrA, Levine et al. (6) proposed that Met residues in pneumoniae (12). The overexpression and purification of the proteins may serve as a ‘‘built-in’’ antioxidant defense system yeast MsrA in E. coli resulted in an Ϸ24 kDa (Fig. 1) protein to protect proteins from oxidation under conditions of oxida- by analysis on an SDS͞PAGE. The molecular mass of the tive stress. This concept is consistent with the results presented recombinant MsrA was slightly higher than the predicted here. Overall, MsrA exerts its protection against oxidative Ϸ21-kDa protein, partly attributable to the six histidine resi- stress by maintaining a low level of oxidized Met, either in dues fused to its N terminus. In general, the characteristics of protein-bound or free amino acid form; thus, in effect it serves the yeast MsrA enzyme activities toward Met(O) were the as an antioxidant. Also, by keeping the level of protein Met(O) same as that from cow and E. coli. Like the previously down, MsrA decreases the need for degradation of Met(O)- described enzymes, the yeast MsrA can reduce Met(O) to Met rich proteins and their replacement by de novo synthesis. The either in its protein-bound or free form (Table 1). In addition, required levels of free Met needed for protein synthesis is extract of yeast msrA null mutant showed no reducing activity maintained also by salvaging free Met from free Met(O) by the Downloaded by guest on September 25, 2021 Biochemistry: Moskovitz et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9589

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