From the Department of Chemistry, Indiana University, Bloomington, Indiana 47405
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ABSTRACT |
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Protein tyrosine phosphatases (PTPases) are essential proteins in many cellular processes. In vitro selection was used to evolve high affinity RNA aptamers to the Yersinia PTPase from two random pools varying in length. Selected aptamers from the two different pools share a 21-residue conserved sequence. They bind to their target with dissociation constants of 18 and 28 nM and inhibit the enzyme with IC50 values of 10 and 35 nM, but do not bind a related PTPase. Modification of the PTPase's active site cysteine with the alkylating agent iodoacetate results in a loss of binding affinity. These experiments suggest that the selected aptamers act by binding at or near the active site and might therefore be useful in defining the interactions between PTPases and their targets.
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INTRODUCTION |
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Approximately 30% of intracellular proteins are phosphoproteins. The phosphorylation and dephosphorylation of these phosphoproteins regulate a variety of metabolic processes, including signal transduction, growth, differentiation, and viral infection (1-3). The phosphorylation states of proteins are in turn controlled by a variety of protein kinases and phosphatases. Together, protein kinases and phosphatases are estimated to encode for as much as 4% of the eukaroytic genome (4).
While the number and complexity of protein kinases have been
appreciated for many years, the importance of protein phosphatases has
emerged more recently. In particular, protein tyrosine phosphatases (PTPases)1 seem to play a
critical role in regulating cellular and viral replication. Over 75 PTPases have been identified to date, and based on genome
sequencing estimates, there are expected to be over 500 in humans alone
(5). The PTPase catalytic domain consists of roughly 250 amino acids
and is the only region of sequence similarity among the multiple,
different PTPases (3, 6). The PTPase active site contains a
catalytically essential cysteine (7-10) embedded within a highly
conserved, 11-residue sequence motif
((Ile/Val)-His-Cys-X-Ala-Gly-X-Gly-Arg-X-Gly).
A subclass of protein phosphatases that can dephosphorylate serine,
threonine, and tyrosine residues has also been identified. These
so-called dual-specificity phosphatases are marked by the active site
sequence His-Cys-X-X-Gly-X-X-Arg-Ser-(Thr). The
crystal structures of the Yersinia PTPase, Yop51*162, the
human PTPase, PTP1B, and the dual-specificity phosphatase, VHR, have
all been solved (11-13). All three enzymes contain a phosphate-binding
loop with the catalytic cysteine residue at its center. The crystal
structures of two receptor tyrosine phosphatases, RPTP
D1 and
RPTPµ D1, have also recently been determined (14, 15). The secondary
and tertiary structures of the catalytic domains of the receptor
tyrosine phosphatases are largely similar to those of Yop51*
162 and
PTP1B, and they also appear to contain phosphate-binding loops at their
active sites.
Since PTPases are emerging as important proteins in oncogenesis and pathogenesis, they may prove to be good targets for drug discovery. Many of the PTPases show excellent substrate specificity; interactions with substrates are determined by the amino acids flanking the phosphorylated tyrosine target (16-19). Unfortunately, the development of specific inhibitors has proven to be difficult. Most of the known inhibitors to PTPases (vanadate, iodoacetate) act by general modification of common active site residues. Even peptide-based inhibitors that take advantage of PTPase recognition "codes" do not always possess specificity toward only one PTPase.
In vitro selection can be used to generate nucleic acid
binding species (aptamers) that bind tightly to protein targets,
including those that are not normally thought to bind nucleic acids
(reviewed in Osborne and Ellington (20)). Anti-protein aptamers have
been shown to bind their targets with extremely high specificity. For example, aptamers that target the II isozyme of protein kinase C do
not recognize the
isozyme, which is 80% identical (21). Similarly,
aptamers that recognize basic fibroblast growth factor do not recognize
acidic fibroblast growth factor (22, 23), despite the fact that the two
proteins are 55% identical (24).
Since PTPases present a phosphate-binding loop to their substrates, we hypothesized that this class of enzymes might productively interact with other phosphate-laden compounds, such as nucleic acids, and thus might prove to be excellent targets for in vitro selection experiments. If anti-PTPase aptamers could be selected, they might be expected to localize to the protein active site and be highly specific inhibitors of enzymatic activity. To test this hypothesis, we focused on one of the best studied of the PTPases, the bacterial enzyme Yop51. The gene encoding Yop51 has been found to be a virulence determinant in the genus Yersinia (25, 26), which includes the causative agent of the bubonic plague (7). The kinetic parameters (27-31), substrate specificity (17, 19, 32), and structure (11, 33, 34) of the Yop51 enzyme have been determined. We have selected aptamers that bind tightly and specifically to Yop51. As predicted, the aptamers localize to the protein active site and inhibit enzymatic activity. The aptamers selected to bind Yop51 or other PTPase targets should prove useful for studying protein-protein interactions, dissecting the complex web of cellular signal transduction pathways, and developing novel pharmaceuticals.
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EXPERIMENTAL PROCEDURES |
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Materials--
Yop51*162 was prepared in the laboratory of
Dr. Dixon at the University of Michigan according to previously
published protocols (27). Yop51* was purchased from New England Biolabs
(Beverly, MA). [
-32P]ATP and
[
-32P]UTP were from NEN Life Science Products. All
other chemicals were reagent grade or molecular biology grade.
Pools--
Selection experiments that targeted Yop51*162 were
initiated with two different RNA pools, N30 and N71, whose synthesis
and purification has previously been described (35, 36). In short, both
pools contained randomized regions of either 30 or 71 nucleotides, flanked by constant regions that were required for enzymatic
amplification. The 3'- constant region allowed cDNAs to be
synthesized from selected RNAs. Both constant regions were utilized
during polymerase chain reaction amplification, and the 5'-constant
region contained a T7 promoter sequence which allowed RNA molecules to
be regenerated from double-stranded DNA templates. The double-stranded
DNA pools (1 µg; 2 × 1013 sequences) served as
templates for in vitro transcription; transcripts were
generated using an Ampliscribe kit (Epicentre Technologies, Madison,
WI) according to the manufacturer's instructions. Following transcription, the RNA pools were purified on 10% denaturing
polyacrylamide gels, and the amount of RNA isolated was quantitated
based on an extinction coefficient of 0.025 µg ml
1
cm
1. In the first round of selection approximately 9 × 1013 N30 RNA molecules were used, and approximately
6.5 × 1013 N71 RNA molecules were used. In other
words, almost all sequences in the original double-stranded DNA pool
should have been represented several times over.
In Vitro Selection--
The concentration of Yop51*162 in
each round was 0.05 µM, while the concentration of the
N30 RNA pool was 0.76 µM, and the concentration of the
N71 RNA pool was 0.54 µM. RNA pools in selection buffer
(20 mM Tris (pH 7.6), 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol; 100 µl final volume) were thermally equilibrated by heating to 65 °C
for 3 min and cooling to room temperature over a period of 10 min. To
exclude filter-binding sequences from the selection, the thermally
equilibrated pools were first passed over a 0.45-µm HAWP filter
(Millipore, Bedford, MA) and washed with an equal volume (100 µl) of
selection buffer. Enough Yop51*
162 was then added to the eluates
(200 µl) to achieve a final concentration of 0.05 µM.
To increase the stringency of rounds 6-8 of the selection with the N30
RNA pool, a nonspecific competitor, tRNA (0.02 µM), was
pre-equilibrated with Yop51*
162 at room temperature for 10 min prior
to initiation of the binding reaction. The binding reactions were
incubated at room temperature for 60 min, vacuum-filtered over a HAWP
filter at 5 p.s.i., and washed once with 2.5 volumes of selection
buffer. The filters were eluted twice with 200 µl of 7 M
urea, 100 mM sodium citrate (pH 5.0), 3 mM EDTA
for 5 min at 100 °C, and the nucleic acids in the eluate were
precipitated with ethanol. The eluted RNA pools were resuspended in 25 µl of water, and 10 µl were reverse-transcribed in 50 mM Tris (pH 8.0), 40 mM KCl, 6 mM
MgCl2, 0.8 mM dNTPs, 2.5 units of avian
myeloblastosis virus reverse transcriptase (Seikagaku, Ijamsville, MD)
(20 µl total volume) for 45 min at 42 °C. A portion of the reverse
transcription reaction (10 µl) was then combined with 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM
MgCl2, 5% acetamide, 0.1% Nonidet P-40, 0.2 mM dNTPs, 2.5 units of Taq polymerase (90 µl;
100 µl final volume) for the N30 pool and 30 mM Tricine
(pH 8.3), 50 mM potassium acetate, 0.5% Triton X-100, 1.5 mM magnesium acetate, 5% acetamide, 0.2 mM
dNTPs, 2.5 units of Taq polymerase (90 µl; 100 µl final
volume) for the N71 pool. Both pools were amplified using the same
thermal cycle, 94 °C for 45 s, 50 °C for 1 min, 72 °C for
1.5 min. A small portion of the amplification reaction was analyzed by
agarose gel electrophoresis every three polymerase chain reaction
cycles to determine if amplified, double-stranded DNA was present. This precaution guarded against the overamplification of DNA and the subsequent selection of catenated or deleted polymerase chain reaction
products. Following precipitation, two-fifths of the amplified DNA was
used as a template to transcribe the next generation of RNA molecules.
Approximately 20-40 µg of RNA were typically recovered for each
round of selection. Given the initial complexity of the pool, while
some high affinity binding species might have been lost following the
first round of selection, no high affinity binding species should have
been lost in subsequent rounds.
Binding Assays--
After completion of rounds 5 and 8, each
pool was assayed for its ability to bind Yop51*162. RNA pools were
radiolabeled with [
-32P]UTP (3000 Ci/mmol) in a
20-µl transcription reaction containing 83 nM labeled UTP
and 7.5 mM unlabeled UTP and subsequently gel-isolated. The
radiolabeled RNA pools (0.76 µM final concentration) were mixed with Yop51*
162 (0.5 µM final concentration) in
100 µl of selection buffer for 1 h at room temperature. The
reactions were then filtered as described above, and the filters were
exposed to a PhosphorImager plate. The amount of retained radioactivity was determined using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The amount of radioactivity retained on the filter was compared with the amount of radioactivity introduced into the binding reaction to determine percent binding.
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(Eq. 1) |
Cloning and Sequencing--
Each selected pool was ligated into
a TA cloning vector (Invitrogen, Carlsbad, CA) and cloned into
Escherichia coli InvF' (Invitrogen). Plasmid DNA was
isolated and sequenced using Sequenase 2.0 (U. S. Biochemical Corp.,
Cleveland, OH) according to the protocol provided with the enzyme.
Secondary structures for individual aptamers were predicted using the
program Mulfold (39).
Inhibition Studies-- Aptamer inhibition of Yop51* dephosphorylation of a peptide substrate was measured using a tyrosine phosphatase assay system (Promega, Madison, WI). The kit provides a fully phosphorylated peptide substrate (DADE(pY)LIPQQG) and measures the release of phosphate by following the formation of a molybdate-malachite green-phosphate complex. Individual aptamers, ranging in concentration from 0 to 2 µM, were incubated in 25 mM bis-tris propane and 5 mM MgCl2 (50 µl final volume) with 120 µM peptide and 0.2 nM Yop51* for 6 min at ambient temperature. The substrate concentration is higher than the Km for Yop51* under these conditions. The data points that were obtained were within the linear range of the assay. Following complex formation, the amount of phosphate produced was determined by measuring the absorbance of the solution at 600 nm.
Active Site Modification-- To specifically modify the active site cysteine of Yop51*, approximately 3 µg of protein were incubated in 15 µl of selection buffer with 2 mM iodoacetate for 30 min at 37 °C (40). Varying amounts of modified Yop51* were added directly to radiolabeled RNA in selection buffer. The phosphatase reaction was then incubated at ambient temperature for 1 h prior to filtration on the vacuum manifold. As a control, unlabeled RNA was incubated with iodoacetate under identical conditions, repurified on a 10% acrylamide gel, labeled, and incubated with modified and unmodified protein in identical reactions.
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RESULTS AND DISCUSSION |
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Selection of Anti-Yop51* Aptamers--
To determine whether
aptamers that recognized the active site of a protein tyrosine
phosphatase could be isolated, we utilized the tyrosine phosphatase
(Yop51) from Yersinia enterocolitica as a selection target.
The structure of Yop51*162 with a tungstate anion had been
determined and revealed that phosphate was likely bound in a relatively
open, positively charged pocket (11, 33, 34). We hypothesized that the
phosphodiester backbone of a RNA molecule might interact with the
PTPase in a manner similar to that of phosphate, and that particular
RNA sequences or structures would be chemically complementary to the
positively charged pocket.
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A Sequence and Structural Motif That Binds Yop51*-- Surprisingly, the aptamers derived from the N30 pool shared extensive sequence similarity with aptamers derived from the N71 pool. Twenty-one residues in the consensus anti-Yop51* aptamer derived from the N30 pool were identical to the sequence of the consensus anti-Yop51* aptamer derived from the N71 pool (Fig. 1). These results are similar to results of selections carried out against both the human immunodeficiency virus type 1 Rev protein and vascular endothelial growth factor. Anti-Rev aptamers isolated from RNA libraries that spanned either 18 or 32 random sequence positions contained a similar sequence and secondary structural motif (41-43). Similarly, anti-vascular endothelial growth factor aptamers isolated from modified RNA libraries that spanned either 30 or 50 random sequence positions were similar to one another (44).
The selection of similar sequence motifs from pools of different lengths can potentially be attributed to what has been called "the tyranny of short motifs." In other words, a shorter motif has a greater initial chance of survival because it is present in a random sequence pool at a higher frequency than is a longer motif. A longer motif will be preferentially selected only if its affinity for a target is much higher than that of the shorter motif. However, in the current selection it seems unlikely that the commonality of the selected, 21-residue motif was an artifact of prevalence. A particular 21-residue motif will be found roughly once in every 421 or 4 × 1012 sequences. Given that the pools used in the selections that targeted Yop51* contained from 1 to 2 × 1013 unique nucleic acid species, the sequence motif that was eventually returned was almost as complex as possible. Moreover, given that the same motif was derived from different pools with different numbers of random sequence positions and different constant regions, it is more likely that the common anti-Yop51* binding motif is optimal, at least for sequences of this size. The program Mulfold was used to generate possible secondary structures for the aptamers. Interestingly, the aptamers from the N30 selection folded into a simple stem-loop structure (Fig. 2), while the aptamers from the N71 selection appeared to fold into a convoluted structure with many loops (not shown). Given that the same, long sequence motif was identified in both pools, it is unlikely that this motif was being presented in different structural contexts. To examine whether the identified sequence motif might be similarly presented in both sets of aptamers, suboptimal foldings of the aptamers derived from the N71 pool were examined. In several of the suboptimal folds, a stem-loop structure that was very similar to the stem-loop structure predicted for the N30 pool was observed (Fig. 2). It is interesting to note that the N30 aptamers fortuitously use three residues from their constant region to form the paired stem that contributes to the common sequence and structural motif, while in the N71 aptamers the same three residues are derived from the randomized core.
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Anti-Yop51* Aptamers Bind to Yop51* with High Affinity and
Specificity--
Since the degenerate pools had been winnowed to
predominant aptamer families, single members of each selected
population (N30yc5, N71yc2, and N71yc16) were further characterized.
Interactions between these anti-Yop51* aptamers and Yop51*162 were
probed using a filter-binding assay similar to that employed for
selection. When binding was examined as a function of protein
concentration, the dissociation constants of the aptamer-protein
complexes were found to be 28 nM for N30yc5 and 18 nM for N71yc16 (Fig. 3).
Similar experiments carried out with N30yc5 and the full-length
protein, Yop51*, gave a dissociation constant of ~50 nM
(data not shown).
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Anti-Yop51* Aptamers Inhibit Yop51*-- To determine if the anti-Yop51* aptamers might be useful as reagents for probing the function of Yop51 in vitro or in vivo, we examined whether anti-Yop51* aptamers could inhibit tyrosine phosphatase activity. These studies used a phosphorylated peptide based on an autophosphorylation site in the epidermal growth factor receptor (19) as a physiologically relevant substrate. When aptamers N30yc5 and N71yc2 were assayed for their ability to inhibit PTPase activity, the observed IC50 values were 35 and 10 nM, respectively (Fig. 4). These values were consistent with the dissociation constants calculated for aptamers complexed with the truncated protein. No inhibition of enzymatic activity was observed with the unselected RNA pools.
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Anti-Yop51* Aptamers Bind to the Active Site of Yop51*--
To
further test the hypothesis that anti-Yop51* aptamers were binding and
blocking access to the active site of the protein, we specifically
modified the active site of Yop51* and assayed whether or not the
modifications blocked interactions with anti-Yop51* aptamers. It has
previously been shown that iodoacetate labels only the active site
cysteine of Yop51*162 (40). Following treatment with iodoacetate the
enzyme's PTPase activity was completely inhibited, as assayed by
para-nitrophenyl phosphate cleavage. Unmodified and modified
proteins were then mixed with anti-Yop51* aptamers and complexes were
isolated by filtration over modified cellulose, as before. As can be
seen in Fig. 5, alkylation of the Yop51*
active site completely inhibits not only the activity of the protein
but also binding of the RNA aptamers to the protein. To control for the
possibility that residual iodoacetate might modify the structure or the
function of the RNA in some way, the aptamers were incubated with
iodoacetate alone, purified, and incubated with unmodified Yop51*.
Modification of the RNA resulted in little or no loss of the binding
activity (data not shown).
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FOOTNOTES |
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* 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.
Present address: ICMB A4800/MBB 1.206, 26th and Speedway,
University of Texas, Austin, TX 78712.
§ Present address: Dept. of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR.
¶ Biological Chemistry; M5416A MED Sci I, Ann Arbor, MI 48109-0606.
To whom correspondence should be addressed: ICMB A4800/MBB
1.206, 26th and Speedway, University of Texas, Austin, TX 78712. Tel.:
512-232-3424; Fax: 512-471-7014; E-mail:
andy.ellington{at}mail.utexas.edu.
1 The abbreviations used are: PTPase, protein tyrosine phosphatase; bis-tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
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