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Originally published In Press as doi:10.1074/jbc.M307152200 on July 29, 2003

J. Biol. Chem., Vol. 278, Issue 43, 41734-41741, October 24, 2003
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Aurintricarboxylic Acid Blocks in Vitro and in Vivo Activity of YopH, an Essential Virulent Factor of Yersinia pestis, the Agent of Plague*

Fubo Liang{ddagger}§, Zhonghui Huang{ddagger}§, Seung-Yub Lee{ddagger}, Jiao Liang{ddagger}, Maya I. Ivanov¶, Andres Alonso||, James B. Bliska¶, David S. Lawrence**, Tomas Mustelin||, and Zhong-Yin Zhang{ddagger}**{ddagger}{ddagger}

From the Departments of {ddagger}Molecular Pharmacology and **Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, the Department of Molecular Genetics and Microbiology and Center for Infectious Diseases, State University of New York at Stony Brook, New York 11794, and ||The Burnham Institute, La Jolla, California 92037

Received for publication, July 3, 2003 , and in revised form, July 26, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Yersinia are causative agents in human diseases ranging from gastrointestinal syndromes to Bubonic Plague. There is increasing risk of misuse of infectious agents, such as Yersinia pestis, as weapons of terror as well as instruments of warfare for mass destruction. YopH is an essential virulence factor whose protein-tyrosine phosphatase (PTP) activity is required for Yersinia pathogenicity. Consequently, there is considerable interest in developing potent and selective YopH inhibitors as novel anti-plague agents. We have screened a library of 720 structurally diverse commercially available carboxylic acids and identified 26 YopH inhibitors with IC50 values below 100 µM. The most potent and specific YopH inhibitor is aurintricarboxylic acid (ATA), which exhibits a Ki value of 5 nM for YopH and displays 6–120-fold selectivity in favor of YopH against a panel of mammalian PTPs. To determine whether ATA can block the activity of YopH in a cellular context, we have examined the effect of ATA on T-cell signaling in human Jurkat cells transfected with YopH. We show that YopH severely decreases the T-cell receptor-induced cellular tyrosine phosphorylation, ERK1/2 activity, and interleukin-2 transcriptional activity. We demonstrate that ATA can effectively block the inhibitory activity of YopH and restore normal T-cell function. These results provide a proof-of-concept for the hypothesis that small molecule inhibitors that selectively target YopH may be therapeutically useful. In addition, it is expected that potent and selective YopH inhibitors, such as ATA, should be useful reagents to delineate YopH's cellular targets in plague and other pathogenic conditions caused by Yersinia infection.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The genus Yersinia include three species that are pathogenic for rodents and humans: Yersinia pestis is the agent responsible for Bubonic Plague (also known as Black Death), Yersinia enterocolitica produces a broad range of gastrointestinal syndromes, and Yersinia pseudotuberculosis causes adenitis and septicemia (1). Credible estimates of the number of people killed by this bacterium during the course of human history approach 200 million (2). Despite efforts to eradicate the disease, it has recently been recognized by the World Health Organization as a re-emerging public health concern. In addition, several multidrug resistant strains have been found in Y. pestis (3, 4). Moreover, there is increasing risk of misuse of infectious agents, such as Y. pestis, as weapons of terror, as well as instruments of warfare for mass destruction (5–7). Thus, there is an urgent need to devise unique and effective treatments that could be implemented soon after a bioterrorist attack.

All three pathogenic species of Yersinia contain an extrachromosomal plasmid of 70 kb that is essential for virulence (8). This plasmid encodes the genes of a type III secretion system and several of the bacterial virulence factors known as Yops1 (Yersinia outer membrane proteins) (9). The type III secretion system is a highly conserved macromolecular machinery among pathogenic Gram-negative bacteria, that upon contact with a eukaryotic cell injects a set of bacterial effector proteins into the lumen of the target cells. Six Yersinia effectors, YopH, YopE, YopJ/P, YpkA/YopO, YopT, and YopM, have been identified (10). The expression of Yops is correlated with the capacity of the bacterium to avoid host defense mechanisms (11). Once inside the host cell, Yops disrupt signaling cascades that activate the processes of phagocytosis, cytokine production, and respiratory burst, resulting in inhibition of the innate and adaptive immune responses (12).

YopH was identified as an essential and obligatory virulence factor for Yersinia pathogenesis as plasmids that have a nonfunctional yopH gene are avirulent (13–16). Surprisingly, the YopH protein contains amino acid sequences (including the active site sequence HC(X)5R) similar to the eukaryotic protein-tyrosine phosphatase (PTP) superfamily (17). In fact, YopH is the most active PTP characterized to date (18). Because bacteria in general do not contain tyrosine-phosphorylated proteins (19), the requirement of the YopH PTP activity for Yersinia pathogenicity suggests that it mediates a novel mechanism of bacterial pathogenesis. Indeed, production of YopH during Yersinia infection is associated with dephosphorylation of proteins in human epithelial cells and murine macrophages (20–23). The identified celluar targets of YopH include p130cas, focal adhesion kinase p125FAK, and paxillin, all tyrosine-phosphorylated proteins found in the focal adhesion complexes (24, 25). These findings are consistent with the observations that YopH activity causes the disassembly of focal adhesion, which impairs the entry of the bacteria into HeLa cells or their phagocytosis by macrophages and neutrophils (24–27). Thus, the YopH-mediated dephosphorylation of focal adhesion proteins may be important for the ability of Yersinia to escape the host innate immune responses.

Since the acquired immune responses are crucial to the survival of infected animals and all three Yersinia species have a tropism for lymphatic tissue (28–30), it is possible that YopH may also have an effect on components of the adaptive immune system. Indeed, it has been reported that Y. pseudotuberculosis can directly interfere with T and B cell antigen recepter-mediated activation and the lymphocyte inhibitory effects are dependent on YopH (31). The presence of YopH in T and B cells results in hypophosphorylation of almost all tyrosine-phosphorylated components associated with the antigen receptor signaling complex after receptor activation. More recently, YopH was shown to inhibit phosphatidylinositol 3-kinase dependent proliferation and interleukin-2 (IL-2) secretion of stimulated T-lymphocytes infected by Y. enterocolitica (32). Consequently, T-cells transiently exposed to Yersinia are unable to flux calcium and produce cytokines. It appears that YopH can suppress the functions of both T- and B-cells thereby preventing the development of an appropriate adaptive immune response.

It is clear that there are diverse mechanisms for YopH to impair and evade both the innate and adaptive host immunity. It is also evident that the PTP activity of YopH is essential for the bacterial pathogenicity. For example, conversion of the essential cysteine residue to alanine in YopH abolishes the PTP activity in Y. pseudotuberculosis and eliminates the virulence of the bacterium in a murine infection model (20, 21). Because the PTP activity of YopH is essential for the virulence of Yersinia, specific inhibitors targeted to YopH are expected to render the bacteria avirulent. Thus, potent and specific inhibitors for the Yersinia PTP could serve as effective agents to block the spread and proliferation of Y. pestis infection. In this study, we describe the identification and biochemical characterization of a potent and specific small molecule YopH inhibitor, ATA. We further demonstrate that the inhibitory effects of YopH on T-cell signaling can be blocked by ATA, validating the concept that inhibition of YopH may be therapeutically useful.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Materials—ATA was purchased from Sigma. p-Nitrophenyl phosphate (pNPP) was purchased from Fluke Co. All other chemicals and reagents were of the highest grade available commercially. Anti-phosphotyrosine mAb (PY20) was from BD Biosciences (San Diego, CA). Polyclonal anti-ERK1/2 and anti-phospho-ERK1/2 (Thr-202/Tyr-204) antibodies were purchased from Cell Signaling (Beverly, MA). mAb 12CA5 to hemagglutinin was from Roche Applied Science. Monoclonal anti-T-cell receptor (TCR) antibodies C305 were obtained from American Type Culture Collection (Manassas, VA).

Protein Purification—YopH was expressed and purified as described previously (18). The catalytic domain of CD45 (containing both D1 and D2) was expressed and purified as a recombinant GST fusion protein. Other recombinant PTPs, PTP1B, Cdc25A, VHR, T-cell PTP (TCPTP), and HePTP were expressed in Escherichia coli and purified as described previously (33, 34).

Screening of a Carboxylic Acid Library for YopH Inhibitors—A library of 720 structurally diverse commercially available carboxylic acids that differ by molecular weight, charge, polarity, hydrophobicity, and sterics was acquired from Aldrich and Sigma. The compounds were dissolved in N,N-dimethylformamide (DMF) to a concentration of 0.5 M and stored in eight 96-well plates, each plate consisting of 90 compounds. The 6 empty wells were used for control. The compounds were further diluted to 2 mM with DMF.

To screen this carboxylic acid library for YopH inhibitors, the effect of each library member on the YopH-catalyzed pNPP hydrolysis was determined. The YopH-catalyzed hydrolysis of pNPP in the presence of 100 µM carboxylic acid was assayed at 30 °C in a 200-µl reaction system in a 96-well plate. Each reaction contained 10 µl of 2 mM compound in DMF (final concentration 100 µM) and 190 µl of assay buffer (50 mM 3,3-dimethylglutarate, 1 mM EDTA, 1 mM dithiothreitol, pH 7.0 with an ionic strength of 0.15 M adjusted by addition of NaCl) containing 2 mM pNPP and 10 nM YopH. The PTP-catalyzed reaction was started by addition of the enzyme. 10 µl of DMF (i.e. no carboxylic acid) was used as a control. The YopH-catalyzed hydrolysis of pNPP was measured by monitoring the absorbance at 405 nm of the product p-nitrophenol continuously, with a SpectraMAX 340 microplate spectrophotometer (Molecular Devices). The initial rate was obtained by calculating the slope of the product versus the time curve. Compounds that display significant inhibition at 100 µM were subject to IC50 measurements.

IC50 Measurement—The PTP-catalyzed hydrolysis of pNPP in the presence of inhibitor was assayed at 30 °C in a 200-µl reaction system in the same assay buffer described above. At various concentrations of the compound, the initial rate at fixed pNPP concentrations (equal to the corresponding Km values for each PTP) was measured by continuously following the production of p-nitrophenol as described above. The IC50 value was determined by plotting the relative PTP activity toward pNPP versus inhibitor concentration and fitting to Equation 1 using Kaleidagraph.

(Eq. 1)

In this case, Vi is the reaction velocity when the inhibitor concentration is [I], V0 is the reaction velocity with no inhibitor, and IC50 = Ki + Ki[S]/Km. Therefore, when the substrate concentration [S] is equal to Km, IC50 = 2Ki.

Ki Measurement—The YopH PTP-catalyzed hydrolysis of pNPP in the presence of ATA was assayed at 30 °C and in assay buffer described above. The reaction was initiated by addition of the enzyme YopH to a reaction mixture (200 µl) containing various concentrations of pNPP (ranging from 0.2 to 5 Km) and various fixed concentrations of ATA, and quenched by addition of 50 µl of 5 M NaOH. The nonenzymatic hydrolysis of the substrate was corrected by measuring the control without addition of enzyme. The amount of product p-nitrophenol was determined from the absorbance at 405 nm detected by a Spectra MAX340 microplate spectrophotometer using a molar extinction coefficient of 18,000 M–1 cm–1. The inhibition constant and inhibition pattern were evaluated using the program GraFit.

Plasmid Construction, Cell Culturing, and Transfection—The cDNA for YopH was subcloned into the pEF5HA eukaryotic expression vector, which encodes an N-terminal hemagglutinin (HA) epitope tag. Human leukemic Jurkat T-cells expressing SV40 large T antigen (Jurkat-TAg cells) were kept at logarithmic growth in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum and 50 units/ml of penicillin G and 50 µg/ml streptomycin. 20 x 106 cells were transiently transfected with a total of 10 µg of either pEF/HA empty vector or HA-tagged YopH plasmid by electroporation at 800 microfarads and 280 V. 36 h after electroporation, cells were harvested and either left untreated or stimulated with C305 antibodies for 5 h. 200 nM ATA (diluted from 50 mM stock in aqueous solution) was added to cell medium during electroporation or stimulation.

Immunoblotting—Cells were lysed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EGTA, 1 mM Na3VO4, 10 µg/ml aprotinin and leupeptin, 1 mM phenylmethylsulfonyl fluoride. Cell lysates were clarified by centrifugation at 16,000 rpm for 15 min. Protein concentration was determined by the BCA protein assay kit from Pierce. Equal protein amount of cell lysates was then subjected to SDS-PAGE and transferred electrophoretically to a nitrocellulose membrane, which was immunoblotted by appropriate antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. The blots were developed by the enhanced chemiluminescence technique (ECL kit, Amersham Biosciences) according to the manufacturer's instructions.

Luciferase Assays—20 x 106 cells were transfected by electroporation with 2 µg of NFAT/AP-1-luc plasmid, 3 µg pCMV-{beta}-gal together with 5 µg of empty pEF/HA vector or YopH plasmids. Cells were then stimulated with the TCR antibodies for 5 h. 200 nM ATA was added during electroporation or stimulation. Cells were lysed in 100 µl of 100 mM potassium phosphate, pH 7.8, 1 mM dithiothreitol, and 0.2% Triton X-100. The final assay sample contained 50 µl of cell lysate, 100 µl of ATP solution (10 mM ATP, 35 mM glycylglycine, pH 7.8, and 20 mM MgCl2), and 100 µl of luciferin reagent (0.27 mM coenzyme A, 0.47 mM luciferin, 35 mM glycylglycine, pH 7.8, 20 mM MgCl2). The activity was measured in an automatic luminometer (Monolight 3010, Pharmingen, San Diego, CA). The activity of a cotransfected {beta}-galactosidase was measured to normalize the luciferase activity for transfection efficiency.

Macrophage Infections—J774A.1 murine macrophage-like cells were prepared for infection assays as described (35). Three hours prior to infection the cells were incubated in tissue culture medium containing 100 µM ATA. The cells were infected with Y. pseudotuberculosis as described (35) for 1 h in the presence of 100 µM ATA. Protein dephosphorylation was assayed by antiphosphotyrosine immunoblotting as described (35).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
As noted in the Introduction, Y. pestis is an emerging public health threat and a potential weapon in biological warfare and bioterrorism. Because the YopH phosphatase activity is essential for virulence in the Yersinia pathogen, small molecule inhibitors of YopH may constitute a novel family of anti-plague agents. Moreover, inhibition of YopH may also widen the time window in which treatment for the plague with broad-spectrum antibiotics would be effective. However, few potent and selective YopH inhibitors are described. Some of the YopH inhibitors that have been identified to date, such as vanadate and suramin, tend to exhibit broad activity against a multitude of mammalian PTP targets (36). More recently, several divalent and trivalent {alpha}-ketocarboxylic acids and 4-(carboxymethyloxy) phenylalanine containing tripeptides have been discovered that exhibit low micromolar affinity toward YopH (37, 38). Unfortunately, these compounds also inhibit the mammalian PTP1B with similar potency. The goal of this study was to identify potent low molecular weight compounds that can specifically inhibit YopH without affecting host PTPs and block the cellular intoxication by YopH.

The active site-directed phosphotyrosine (pTyr) provides key elements for PTP substrate recognition (39–43). Thus, a common approach for PTP inhibitor design is to develop nonhydrolyzable pTyr surrogates that contain both a phosphate mimic that substitutes the phosphoryl group and an aromatic scaffold that can occupy the active site pocket in a manner reminiscent of the aromatic ring in pTyr (44, 45). Due to the conserved nature of the pTyr-binding pocket, nonhydrolyzable pTyr surrogates usually do not display strong selectivity against different PTPs. However, inhibitor potency and selectivity can be achieved by linking the nonhydrolyzable pTyr mimetic with an appropriate structural element to target both the active site and a unique adjacent site (46).

Because the YopH catalytic domain shares only 20–30% sequence identity with the mammalian PTPs (47), it seems quite feasible to acquire YopH specific inhibitors. Indeed, we have recently identified a specific YopH small molecule inhibitor, p-nitrocatechol sulfate, which exhibits a Ki of 25 µM for YopH and displays a 13–60 fold selectivity in favor of YopH against a panel of mammalian PTPs (48). However, the modest potency and in vivo instability of the sulfate ester of p-nitrocatechol sulfate limit its further biological evaluation.

Identification of YopH Inhibitors from a Carboxylic Acid Library—As a continued effort to search for potent and selective small molecule inhibitors for YopH, we screened a library of 720 structurally diverse commercially available carboxylic acids that differ by molecular weight, charge, polarity, hydrophobicity, and sterics. Our rationale for employing a carboxylic acid library was based on the fact that the majority of the reported nonhydrolyzable pTyr surrogates contain at least one carboxyl group (45). The ability of the library members to inhibit the YopH-catalyzed hydrolysis of p-nitrophenyl phosphate (pNPP) was assessed at 30 °C in pH 7.0 buffer containing 50 mM 3,3-dimethylglutarate, 1 mM EDTA, 1 mM DTT, with an ionic strength of 0.15 M adjusted by addition of NaCl (for details see "Experimental Procedures"). Out of the 720 carboxylic acids, 26 compounds displayed measurable inhibition of the YopH-catalyzed pNPP hydrolysis at 100 µM concentration. They were selected for further characterization. For each compound, the inhibitory efficacy as determined by the IC50 value was measured at a pNPP concentration fixed at the experimentally determined Km for YopH (2.0 mM). Table I lists the structures of the 26 carboxylic acids together with their associated IC50 values for the YopH-catalyzed reaction. As shown in Table I, the hits that were generated against YopH cover a diverse structural space. In addition, the IC50 values for the hits span several orders of magnitude.


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  		TABLE I
Structures and IC50 values of carboxylic acid inhibitors of YopH

The IC50 value was measured at a pNPP concentration fixed at the experimentally determined Km for YopH (2.0 mM).

 

We have also screened the library with several mammalian PTPs. Several compounds, including 2-fluoro-6-(trifluoromethyl)benzoic acid, trans-4-methyl-1-cyclohexanecarboxylic acid, and diiodofluorescein, inhibit YopH, HePTP, PTP1B, and CD45 to a similar extent at 100 µM concentration (data not shown). By contrast, many others, including 4-hydroxy-3-nitrophenylacetic acid, (S)-(–)-indoline-2-carboxylic acid, 9-fluorenone-1-carboxylic acid, 3,7-dihydroxy-2-naphthoic acid, show no measurable inhibitory activity against HePTP, PTP1B, and CD45 at 100 µM. In addition, 3,5-dihydroxy-2-naphthoic acid, aurintricarboxylic acid (ATA), 4,4'-bis(3-carboxy-4-chloroanilino)tritylchloride, lithocholic acid, Chrome Azurol S, and Mordant Orange 1 display at least several-fold selectivity in favor of YopH. Remarkably, ATA inhibits YopH with an IC50 of 10 nM, which makes it the most potent and selective YopH inhibitor identified to date.

Novel Nonhydrolyzable pTyr Mimetics—The active sites of PTPs are highly conserved. In the pTyr-bound form of PTP1B, the terminal non-bridge phosphate oxygens of pTyr form an extensive array of hydrogen bonds with the main chain nitrogens of the PTP signature motif (residues 215–221) and the guanidinium side-chain of Arg-221. The phenyl ring of the pTyr is engaged in hydrophobic interactions with the active site cavity, formed by the nonpolar side-chains of Val-49, Ala-217, Ile-219, and Gln-262, and the aryl side-chains of Tyr-46 and Phe-182, which sandwich the pTyr ring and delineate the boundaries of the pTyr-binding pocket (42). Given the structural similarity of aryl sulfate/sulfonate/phosphonate moieties to pTyr, they are commonly used as nonhydrolyzable pTyr mimetics (44). In addition, a variety of carboxylic acid containing compounds, such as O-malonyltyrosine, cinnamic acid, 3-carboxy-4-(O-carboxymethyl) tyrosine, salicylic acid, benzoic acid, 2-(oxalylamino)-benzoic acid, 5-carboxy-2-naphthoic acid, and {alpha}-ketocarboxylic acid, are also capable of serving as nonhydrolyzable pTyr surrogates (45). The carboxylate-containing compounds are generally favored for therapeutic development due to their more desirable physical properties (e.g. one less negative charge).

Most of the reported nonhydrolyzable pTyr surrogates contain a phosphate mimic attached to a benzene ring. These compounds usually exhibit affinities for PTPs in the low millimolar range, similar to the Km values for pNPP and pTyr (18, 41). Since our initial screen was performed at 100 µM concentration, most of the 26 lead compounds identified from the carboxylic acid library display IC50 values in the low micromolar range (Table I). Kinetic analysis of a number of the lead compounds indicated that they are reversible and competitive YopH inhibitors. Consequently, many of the newly identified structures represent novel pTyr mimetics with unprecedented affinities for YopH.

A closer examination of the structures revealed several interesting features about the new hits, which may explain their increased affinity for YopH. First, 8 out of the 26 compounds (1-pyreneacetic acid, (S)-(–)-indoline-2-carboxylic acid, 9-fluorenone-1-carboxylic acid, 3,7-dihydroxy-2-naphthoic acid, 1-pyrenecarboxylic acid, 4-fluoro-1-naphthoic acid, 3,5-dihydroxy-2-naphthoic acid, and gallocyanine) contain an extended aromatic system. This is consistent with previous observations that naphthyl and polyaromatic derivatives exhibit enhanced affinities for PTP relative to the corresponding single ring compounds (49–53). It appears that the PTP active site possesses considerable plasticity such that substituted and polyaromatic compounds significantly larger than pTyr can be accommodated to gain additional hydrophobic interactions, which is not available to the single phenyl ring compounds.

Second, although several of the novel pTyr mimetics shown in Table I have the carboxylate group located on a single phenyl ring (e.g. lasalocid, 4-hydroxy-3-nitrophenylacetic acid, 3-nitrocinnamic acid, 2-fluoro-6-(trifluoromethyl)benzoic acid, diiodofluorescein, ATA, 4,4'-bis(3-carboxy-4-chloroanilino)tritylchloride, 4-iodobenzoic acid, 2-oxo-6-pentyl-2H-pyran-3-carboxylic acid, Chrome Azurol S, and Mordant Orange 1), they all contain additional substitutions on the ring that should either strengthen the interactions with the active site or provide additional contacts with regions proximal to the active site. Indeed, we note that salicylic acid, a member of the carboxylic library, did not show appreciable YopH inhibition at 100 µM. In fact, it displayed an IC50 of 38 mM against YopH. However, as shown in Table I, addition of an aromatic ring(s) or other substitutions to salicylic acid greatly increases the binding affinity of lasalocid, 3,7-dihydroxy-2-naphthoic acid, 3,5-dihydroxy-2-naphthoic acid, ATA, Chrome Azurol S, and Mordant Orange 1 for YopH. Similarly, salicylic acid is also a weak competitive inhibitor of PTP1B with an inhibition constant of 19.4 mM, but a 320-fold increase in binding affinity is observed when the salicylic acid is linked to a 2-thioxo-1-benzimidazolyl derivative (51).

Thirdly, it appears that aliphatic acids can also be effective PTP inhibitors when attached to an appropriately functionalized aromatic system. Examples include 1-pyreneacetic acid, 4-hydroxy-3-nitrophenylacetic acid, N-(4-nitrobenzoyl)-6-aminocaproic acid, triphenylacetic acid, and 3,3,3-tris(4-chlorophenyl) propionic acid (Table I). Finally, it is interesting to note that no aromatics are present in 3-noradamantanecarboxylic acid, trans-4-methyl-1-cyclohexanecarboxylic acid, and lithocholic acid. Further studies are required to elucidate the binding interactions between these compounds and YopH.

ATA Is the Most Potent and Selective YopH Inhibitor Identified to Date—The most potent YopH inhibitor identified from the carboxylic acid library is ATA, which displays an IC50 of 10 nM for YopH. Over the years, effects of ATA on biological systems have been extensively studied. For example, ATA is an inhibitor of protein-nucleic acid interactions, ribonucleases, topoisomerases, and RNA synthesis (54–56). ATA is reported to have antiapoptotic effect on sympathetic neurons (57). ATA is also capable of stimulating tyrosine phosphorylation in PC12 cells (58) and increasing ErbB4 phosphorylation in human neuroblastoma SH-SY5Y cells (59). More recently, ATA was found to promote survival and increase proliferation of MDA-231 and MCF-7 breast cancer cells by activating the insulin-like growth factor signaling pathways (60, 61). However, it should be noted that the ATA concentrations used in these studies are greater than 200 µM, which is 20,000-fold higher than its IC50 for YopH.

To further characterize the mechanism of YopH inhibition by ATA, we determined the IC50 as a function of YopH and ATA preincubation time. The results showed that ATA is a reversible YopH inhibitor. The inhibition constant and the mode of inhibition were determined by steady-state kinetic analysis of the YopH with 8 different pNPP substrate concentrations and 4 different ATA concentrations. As shown in Fig. 1, the effect of ATA on the YopH-catalyzed pNPP hydrolysis displayed the characteristic intersecting line pattern for competitive inhibition. The Ki value for the inhibition of the YopH reaction by ATA is 5.0 ± 0.3 nM. This agrees well with the IC50 value determined at the substrate Km. When an inhibitor binds reversibly and competitively, as ATA does to YopH, the IC50 value determined when the substrate concentration is equal to the Km, is twice the Ki value as shown under "Experimental Procedures."



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  		FIG. 1.
Effect of ATA on the YopH-catalyzed pNPP hydrolysis. The Lineweaver-Burk plot displayed the characteristic intersecting line pattern, consistent with competitive inhibition. The experiment was performed at 30 °C and pH 7.0. ATA concentrations were 0 ({circ}), 4 (•), 8 ({square}), and 12 nM ({blacksquare}), respectively.

 

To determine if ATA is specific for YopH, its inhibitory activity toward a panel of mammalian PTPs including PTP1B, TCPTP, HePTP, and CD45, and the dual specificity phosphatases, Cdc25A and VHR, was measured. The IC50 values for each PTPs were determined at a fixed pNPP concentration equal to the Km for each PTPs. Therefore, all of the IC50 values reported in this study directly reflect the affinity of the compound for the enzymes tested. As shown in Table II, ATA displays an IC50 of 10 nM against YopH, while it is much less effective against mammalian PTPs, with an IC50 value of 61 nM for PTP1B, 250 nM for HePTP and CD45, 400 nM for TCPTP, 1.2 µM for VHR, and 1.0 µM for Cdc25A. Thus, ATA is highly selective for YopH, exhibiting a 6 to 120-fold preference for YopH versus all PTPs examined. Together, the results show that ATA is the most potent and specific YopH inhibitor reported to date. It is also of interest to note that although PTP1B and TCPTP share greater than 75% sequence identity within the catalytic domain and have virtually identical pTyr binding pocket (62), the IC50 values of ATA for the two PTPs differ by nearly 7-fold.


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  		TABLE II
Selectivity of ATA against a panel of mammalian PTPs

All measurements were performed at pH 7 and 30 °C, using pNPP as a substrate. The ATA IC50 values for each PTP were measured at fixed pNPP concentrations equal to the corresponding Km values for each PTP. The pNPP Km values for the PTPs are: 2.0 mM for YopH, 2.0 mM for PTP1B, 2.3 mM for TCPTP, 7.7 mM for HePTP, 4.9 mM for CD45, 5.7 mM for VHR, and 12 mM for Cdc25A.

 

Assessment of the Ability of ATA to Block YopH Activity in Vivo—Our ultimate goal is to develop potent and specific YopH inhibitors as anti-plague agents. Given the extraordinary potency and selectivity of ATA toward YopH, we proceeded to evaluate its in vivo efficacy in several cell lines. It has been shown that YopH can block early tyrosine phosphorylation events and suppress cytokine production in T-cells, thereby preventing the development of an adaptive immune response (31, 32). One would predict that inhibition of YopH activity should restore T-cell receptor (TCR) signaling and tyrosine phosphorylation. Since the biochemistry of antigen receptor signaling is well characterized in the human Jurkat T-cell line (63), and the Jurkat cells are easily amendable to transfection, we initially chose this cell line as an experimental system.

We evaluated whether the induction of the tyrosine phosphorylation cascade and ERK1/2 activation in response to TCR cross-linking by the mAb C305 is altered when Jurkat cells are transfected with an HA-tagged YopH construct. As shown in Fig. 2 (lanes 1 and 2), stimulation of Jurkat cells with the C305 antibodies led to activation of early signaling events as evidenced by the increased total protein tyrosine phosphorylation and activation of ERK1/2. As a control, treatment of Jurkat cells with 200 nM ATA had no significant effect on both total protein tyrosine phosphorylation as well as ERK1/2 activation (lanes 3 and 4; however, see paragraph below). When plasmids containing the HA-tagged YopH were transfected into Jurkat cells by electroporation, we observed almost complete annihilation of the C305 induced T-cell activation (lanes 5 and 6), which is consistent with earlier results (31). Similar levels of YopH protein were observed in the transfected Jurkat cells as indicated by anti-HA immunoblotting.



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  		FIG. 2.
Effect of extracellularly applied ATA on T-cell activation in the presence and absence of YopH. Jurkat cells were transiently transfected with HA-tagged YopH plasmid (lanes 5, 6, 7, and 8) or with pEF/HA empty vector (lanes 1, 2, 3, and 4) by electroporation. 36 h after the transfection, the cells were treated with (lanes 3, 4, 7, and 8) or without (lanes 1, 2, 5, and 6) 200 nM ATA for 1 h. Subsequently, the cells were stimulated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) the C305 antibodies in the presence or absence of ATA for 5 h. Cell lysates (20 µg of total protein) were subjected to SDS-PAGE and transferred to nitrocellulose membrane. Tyrosine-phosphorylated proteins, ERK1/2 activation, ERK1/2 protein and HA-tagged YopH were detected by specific antibodies, respectively.

 

We anticipated that ATA would not be cell permeable due to its polyanionic structure. Indeed, others have noted that ATA may not be able to cross the cell membrane (60). We confirmed this expected behavior in two different cell lines. First, we found that 200 nM ATA exerts little effect on the inhibition of T-cell activation by YopH (Fig. 2, lanes 7 and 8) in YopH-transfected Jurkat cells. In addition, we evaluated the effect of ATA in a murine macrophage-like cell line, J774A.1, in which a number of host proteins are dephosphorylated by YopH upon Yersinia infection (21, 35). The macrophages were pretreated with ATA for three hours and then infected with Y. pseudotuberculosis in the presence of ATA. ATA treatment did not lead to any measurable decrease in dephosphorylation of host proteins by YopH (data not shown).

In order to provide the crucial proof-of-concept evidence that inhibition of YopH is therapeutically useful, we sought to introduce ATA inside the cell by electroporation in order to assess the ability of ATA to block YopH activity in vivo. As shown in Fig. 3, when Jurkat cells were stimulated with the anti-TCR antibody C305, there was a dramatic increase in tyrosine phosphorylation and ERK1/2 activation (lanes 1 and 2). Electroporation of ATA (200 nM in the medium) into Jurkat cells had no significant effect on tyrosine phosphorylation and ERK1/2 activation under both stimulated and unstimulated conditions (lanes 3 and 4). This is an important control because several mammalian PTPs have been shown to play important regulatory roles in many different aspects of T-cell physiology (64). Again, full repression of C305-stimulated tyrosine phosphorylation and ERK1/2 activity were observed upon transfection of YopH into Jurkat T-cells (lanes 5 and 6). As expected, introduction of ATA into YopH transfected Jurkat cells by electroporation almost completely restored the C305-stimulated tyrosine phosphorylation level and ERK1/2 activity to the control in the absence of YopH, while ATA exerted little effect on unstimulated cells (lanes 7 and 8). The results support the notion that the phosphatase activity of YopH is responsible for the suppression of T-cell signaling and inhibition of YopH activity restores T-cell function. To provide further evidence for the importance of YopH phosphatase activity on TCR signaling, we also examined the effect of a catalytically impaired mutant YopH, D356A (47). No appreciable tyrosine dephosphorylation and ERK1/2 inactivation were observed in Jurkat cells transfected with YopH/D356A (lanes 9 and 10), due to the lack of YopH phosphatase activity (mimicking nearly 100% YopH inhibition). Collectively, the data suggest that the restoration of TCR signaling in YopH-transfected cells is due to the inhibition of YopH phosphatase activity by ATA.



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  		FIG. 3.
ATA electroporated into Jurkat cells significantly restores the YopH-suppressed TCR signaling. Jurkat cells were transiently transfected with either pEF/HA empty vector (lanes 1–4), HA-tagged wild-type YopH plasmid (lanes 5–8) or a YopH/D356A (DA) mutant (lanes 9 and 10) together with (lanes 3, 4, 7, and 8) or without (lanes 1, 2, 5, 6, 9, and 10) 200 nM ATA by electroporation. Cells were either left unstimulated (lanes 1, 3, 5, 7, and 9) or stimulated (lanes 2, 4, 6, 8, and 10) with C305 antibodies for 5 h as described. Cell lysates of 20 µg of total protein was subjected to SDS-PAGE and transferred to nitrocellulose membrane. Tyrosine-phosphorylated proteins, activated ERK1/2, ERK1/2 protein, and HA-tagged YopH were detected by specific antibodies, respectively.

 

Finally, we also determined the effect of YopH and ATA on the activity of a luciferase reporter gene driven by a tandem NFAT/AP-1 element taken from the interleukin-2 gene promoter as a functional readout for T-cell activation and proliferation. Jurkat cells were electroporated with NFAT/AP-1-luc reporter plasmids, empty pEF/HA vector or YopH plasmids together with or without 200 nM ATA. Subsequently, the cells were treated with or without the TCR mAb C305 followed by measurement of luciferase activity. As shown in Fig. 4, nearly 8-fold increase in NFAT/AP-1 luciferase activity was observed when Jurkat cells were stimulated with C305 antibodies. Again, introduction of ATA by electroporation into cells had no significant effect on the luciferase activity. Transfection of YopH into the cells abolished more than 80% of C305 stimulated NFAT/AP-1 transcriptional activity, while cotransfection of ATA together with YopH elevated the luciferase activity to 80% of the control. Together, these experiments show that potent and selective YopH inhibitors such as ATA can block the inhibitory effect of YopH in human Jurkat T-cells.



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  		FIG. 4.
ATA electroporated into Jurkat cells significantly restores the YopH-blocked NFAT/AP-1 luciferase activation in TCR signaling. Jurkat cells were electroporated with 2 µg of NFAT/AP-1-luc plasmid, 3 µg of pCMV-{beta}-gal, 5 µg of empty pEF/HA vector or YopH plasmids together with or without 200 nM ATA. Subsequently, the cells were stimulated with or without the TCR mAb C305 as described. The activity of the luciferase reporter was measured in an automatic luminometer and expressed in arbitrary units. The activity of cotransfected {beta}-galactosidase was used to normalize transfection efficiency. The data represent the mean of duplicate samples of three independent experiments. S.D. are depicted by error bars.

 


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
The YopH phosphatase activity is essential for virulence in the Yersinia pathogen, which causes diseases such as the plague. Thus, potent and selective YopH inhibitors are expected to serve as novel anti-plague agents. By screening a library of 720 structurally diverse commercially available carboxylic acids, we have identified several compounds that not only possess potent inhibitory activity against YopH but which also display significant selectivity for YopH. Among these, ATA is the most potent and specific small molecule YopH inhibitor, which exhibits a Ki value of 5 nM for YopH and displays 6–120-fold selectivity in favor of YopH against a panel of mammalian PTPs. To determine whether ATA can block the activity of YopH in a cellular context, we have examined the effect of ATA on T-cell signaling in Jurkat cells transfected with HA-tagged YopH. We show that YopH can severely block the TCR-induced cellular tyrosine phosphorylation, ERK1/2 activation, and NFAT/AP-1 reporter gene luciferase activity. We demonstrate that the YopH inhibitory effects can be reversed by the addition of ATA. These results provide a proof-of-concept for the hypothesis that small molecule inhibitors that selectively target YopH may be therapeutically useful. YopH inhibitors may serve as novel antiplague agents. They could also be given in conjugation with broad-spectrum antibiotics so that a maximal window of treatment can be created to treat patients with the plague. Finally, It is expected that novel, tight binding and specific YopH inhibitors should be helpful in the delineation of YopH's cellular targets during the bacterial pathogenesis process.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants AI43389 (to J. B. B.) and AI48506 and the G. Harold and Leila Y. Mathers Charitable Foundation (to Z.-Y. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

{ddagger}{ddagger} An Irma T. Hirschl Career Scientist. To whom correspondence should be addressed. Tel.: 718-430-4288; Fax: 718-430-8922; E-mail: zyzhang{at}aecom.yu.edu.

1 The abbreviations used are: Yop, Yersinia outer membrane protein; ATA, aurintricarboxylic acid; DMF, N,N-dimethylformamide; IL-2, interleukin-2; pNPP, p-nitrophenyl phosphate; PTP, protein-tyrosine phosphatase; pTyr, phosphotyrosine; TCR, T-cell receptor; HA, hemagglutinin; mAb, monoclonal antibody. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Chi-wing Chow and Teddy Yang, Department of Molecular Pharmacology, Albert Einstein College of Medicine, for help in measuring the luciferase activity.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 

  1. Bulter, T. (1985) in Textbook of Medicine (Wyngaarden, J. B., and Smith, L. H., eds) pp. 1600–1603, Saunders, Philadelphia, PA
  2. Brubaker, R. R. (1991) Clin. Microbiol. Rev. 4, 309–324[Abstract/Free Full Text]
  3. Galimand, M., Guiyoule, A., Gerbaud, G., Rasoamanana, B., Chanteau, S., Carniel, E., and Courvalin, P. (1997) N. Engl. J. Med. 337, 677–680[Free Full Text]
  4. Guiyoule, A., Gerbaud, G., Buchrieser, C., Galimand, M., Eahalison, L., Chanteau, S., Courvalin, P., and Carniel, E. (2001) Emerg. Infect. Dis. 7, 43–48[Medline] [Order article via Infotrieve]
  5. McGovern, T. W., Christopher, G. W., and Eitzen, E. M. (1999) Arch. Dermatol. 135, 311–322[Abstract/Free Full Text]
  6. Inglesby, T. V., Dennis, D. T., Henderson, D. A., Bartlett, J. G., Ascher, M. S., Eitzen, E., Fine, A. D., Friedlander, A. M., Hauer, J., Koerner, J. F., Layton, M., McDade, J., Osterholm, M. T., O'Toole, T., Parker, G., Perl, T. M., Russell, P. K., Schoch-Spana, M., and Tonat, K. (2000) J. Am. Med. Assoc. 283, 2281–2290[Abstract/Free Full Text]
  7. Hawley, R. J., and Eitzen, E. M., Jr. (2001) Annu. Rev. Microbiol. 55, 235–253[CrossRef][Medline] [Order article via Infotrieve]
  8. Portnoy, D. A., Wolf-Watz, H., Bolin, I., Beeder, A. B., and Falkow, S. (1984) Infect. Immun. 43, 108–114[Abstract/Free Full Text]
  9. Cornelis, G. R. (1998) Folia Microbiol. 43, 253–261[CrossRef]
  10. Juris, S. J., Shao, F., and Dixon, J. E. (2002) Cell. Microbiol. 4, 201–211[CrossRef][Medline] [Order article via Infotrieve]
  11. Cornelis, G. R., Boland, A., Boyd, A. P., Geuijen, C., Iriarte, M., Neyt, C., Sory, M. P., and Stainier, I. (1998) Microbiol. Mol. Biol. Rev. 62, 1315–1352[Abstract/Free Full Text]
  12. Cornelis, G. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8778–8783[Abstract/Free Full Text]
  13. Bolin, I., and Wolf-Watz, H. (1988) Mol. Microbiol. 2, 237–245[Medline] [Order article via Infotrieve]
  14. Michielis, T., and Cornelis, G. (1988) Microbial. Pathogen 5, 449–459[CrossRef]
  15. Rosqvist, R., Bolin, I., and Wolf-Watz, H. (1988) Infect. Immun. 56, 2139–2143[Abstract/Free Full Text]
  16. Straley, S. C., and Bowmer, W. S. (1986) Infect. Immun. 51, 445–454[Abstract/Free Full Text]
  17. Guan, K. L., and Dixon, J. E. (1990) Science 249, 553–556[Abstract/Free Full Text]
  18. Zhang, Z.-Y., Clemens, J. C., Schubert, H. L., Stuckey, J. A., Fischer, M. W. F., Hume, D. M., Saper, M. A., and Dixon, J. E. (1992) J. Biol. Chem. 267, 23759–23766[Abstract/Free Full Text]
  19. Foster, R., Thorner, J., and Martin, G. S. (1989) J. Bacteriol. 171, 272–279[Abstract/Free Full Text]
  20. Bliska, J. B., Guan, K. L., Dixon, J. E., and Falkow, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 1187–1191[Abstract/Free Full Text]
  21. Bliska, J. B., Clemens, J. C., Dixon, J. E., and Falkow, S. (1992) J. Exp. Med. 176, 1625–1630[Abstract/Free Full Text]
  22. Hartland, E. L., Green, S. P., Phillips, W. A., and Robins-Browne, R. M. (1994) Infect. Immun. 62, 4445–4453[Abstract/Free Full Text]
  23. Andersson, K., Carballeira, N., Magnusson, K.-E., Persson, C., Stendahl, O., Wolf-Watz, H., and Fallman, M. (1996) Mol. Microbiol. 20, 1057–1069[Medline] [Order article via Infotrieve]
  24. Black, D. S., and Bliska, J. B. (1997) EMBO J. 16, 2730–2744[CrossRef][Medline] [Order article via Infotrieve]
  25. Persson, C., Carballeira, N., Wolf-Watz, H., and Fallman, M. (1997) EMBO J. 16, 2307–2318[CrossRef][Medline] [Order article via Infotrieve]
  26. Ruckdeschel, K., Roggenkamp, A., Schubert, S., and Heesemann, J. (1996) Infect. Immun. 64, 724–733[Abstract]
  27. Black, D. S., Montagna, L. G., Zitsmann, S., and Bliska, J. B. (1998) Mol. Microbiol. 29, 1263–1274[CrossRef][Medline] [Order article via Infotrieve]
  28. Autenrieth, I. B., Vogel, U., Preger, S., Heymer, B., and Heesemann, J. (1993) Infect. Immun. 61, 2585–2595[Abstract/Free Full Text]
  29. Autenrieth, I. B., and Firsching, R. (1996) J. Med. Microbiol. 44, 285–294[Abstract/Free Full Text]
  30. Autenrieth, I. B., Hantschmann, P., Heymer, B., and Heesemann, J. (1993) Immunobiology 187, 1–16[Medline] [Order article via Infotrieve]
  31. Yao, T., Mecsas, J., Healy, J. I., Falkow, S., and Chien, Y. (1999) J. Exp. Med. 190, 1343–1350[Abstract/Free Full Text]
  32. Sauvonnet, N., Lambermont, I., van der Bruggen, P., and Cornelis, G. R. (2002) Mol. Microbiol. 45, 805–815[CrossRef][Medline] [Order article via Infotrieve]
  33. Shen, K., Keng, Y.-F., Wu, L., Guo, X.-L., Lawrence, D. S., and Zhang, Z.-Y. (2001) J. Biol. Chem. 276, 47311–47319[Abstract/Free Full Text]
  34. Zhou, B., and Zhang, Z.-Y. (2002) J. Biol. Chem. 277, 13889–13899[Abstract/Free Full Text]
  35. Black, D. S., Marie-Cardine, A., Schraven, B., and Bliska, J. B. (2000) Cell Microbiol. 2, 401–414[CrossRef][Medline] [Order article via Infotrieve]
  36. Zhang, Y.-L., Keng, Y.-F., Zhao, Y., Wu, L., and Zhang, Z.-Y. (1998) J. Biol. Chem. 273, 12281–12287[Abstract/Free Full Text]
  37. Chen, Y. T., and Seto C. T. (2002) J. Med. Chem. 45, 3946–3952[CrossRef][Medline] [Order article via Infotrieve]
  38. Lee, K, Gao, Y., Yao, Z.-J., Phan, J., Wu, L., Liang, J., Waugh, D., Zhang, Z.-Y., and Burke, T. R. Jr. (2003) Bioorg. Med. Chem. Lett. 13, 2577–2581[CrossRef][Medline] [Order article via Infotrieve]
  39. Zhang, Z.-Y., Maclean, D., Thieme-Sefler, A. M., Roeske, R. W., and Dixon J. E. (1993) Anal. Biochem. 211, 7–15[CrossRef][Medline] [Order article via Infotrieve]
  40. Zhang, Z.-Y., Maclean, D., Thieme-Sefler, A. M., McNamara, D., Dobrusin, E. M., Sawyer, T. K., and Dixon, J. E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 4446–4450[Abstract/Free Full Text]
  41. Zhang, Z.-Y., Maclean, D., McNamara, D. J., Sawyer, T. K., and Dixon, J. E. (1994) Biochemistry 33, 2285–2290[CrossRef][Medline] [Order article via Infotrieve]
  42. Jia, Z., Barford, D., Flint, A. J., and Tonks, N. K. (1995) Science 268, 1754–1758[Abstract/Free Full Text]
  43. Sarmiento, M., Puius, Y. A., Vetter, S. W., Keng, Y.-F., Wu, L., Zhao, Y., Lawrence, D. S., Almo, S. C., and Zhang, Z.-Y. (2000) Biochemistry 39, 8171–8179[CrossRef][Medline] [Order article via Infotrieve]
  44. Burke, T. R. Jr., and Zhang, Z.-Y. (1998) Biopolymers 47, 225–241[CrossRef][Medline] [Order article via Infotrieve]
  45. Zhang, Z.-Y. (2003) in Handbook of Cellular Signaling (Bradshaw, R., and Dennis, E., eds) pp. 677–684, Academic Press, New York
  46. Zhang, Z.-Y. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 209–234[CrossRef][Medline] [Order article via Infotrieve]
  47. Zhang, Z.-Y., Wang, Y., and Dixon, J. E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 1624–1627[Abstract/Free Full Text]
  48. Sun, J.-P., Wu, L., Fedorov, A. A., Almo, S. C., and Zhang, Z.-Y. (2003) J. Biol. Chem. 278, 33392–33399[Abstract/Free Full Text]
  49. Kole, H. K., Smyth, M. S., Russ, P. L., and Burke, T. R., Jr. (1995) Biochem. J. 311, 1025–1031[Medline] [Order article via Infotrieve]
  50. Burke, T. R., Jr., Ye, B., Yan, X., Wang, S., Jia, Z., Chen, L., Zhang, Z.-Y., and Barford, D. (1996) Biochemistry 35, 15989–15996[CrossRef][Medline] [Order article via Infotrieve]
  51. Sarmiento, M., Wu, L., Keng, Y.-F., Song, L., Luo, Z., Huang, Z., Wu, G.-Z., Yuan, A. K., and Zhang, Z.-Y. (2000) J. Med. Chem. 43, 146–155[CrossRef][Medline] [Order article via Infotrieve]
  52. Ibrahimi, O. A., Wu, L., Zhao, K., and Zhang, Z.-Y. (2000) Bioorg. Med. Chem. Lett. 10, 457–460[CrossRef][Medline] [Order article via Infotrieve]
  53. Iversen, L. F., Andersen, H. S., Branner, S., Mortensen, S. B., Peters, G. H., Norris, K., Olsen, O. H., Jeppesen, C. B., Lundt, B. F., Ripka, W., Moller, K. B., and Moller, N. P. (2000) J. Biol. Chem. 275, 10300–10307[Abstract/Free Full Text]
  54. Gonzalez, R. G., Haxo, R. S., and Schleich, T. (1980) Biochemistry 19, 4299–4303[CrossRef][Medline] [Order article via Infotrieve]
  55. Sarma, M. H., Feman, E. R., and Baglioni, C. (1976) Biochim. Biophys. Acta, 418, 29–38[Medline] [Order article via Infotrieve]
  56. Hallick, R. B., Chelm, B. K., Gray, P. W., and Orozio, E. W. (1977) Nucleic Acids Res. 4, 3055–3064[Abstract/Free Full Text]
  57. Batistatou, A., and Greene, L. A. (1991) J. Cell Biol. 115, 461–471[Abstract/Free Full Text]
  58. Okada, N., and Koizumi, S. (1995) J. Biol. Chem. 270, 16464–16469[Abstract/Free Full Text]
  59. Okada, N., and Koizumi, S. (1997) Biochem. Biophys. Res. Commun. 230, 266–269[CrossRef][Medline] [Order article via Infotrieve]
  60. Beery, R., Haimsohn, M., Wertheim, N., Hemi, R., Nir, U., Karasik, A., Kanety, H., and Geier, A. (2001) Endocrinology 142, 3098–3107[Abstract/Free Full Text]
  61. Haimsohn, M., Beery, R., Karasik, A., Kanety, H., and Geier, A. (2002) Endocrinology 143, 837–845[Abstract/Free Full Text]
  62. Iversen, L. F., Moller, K. B., Pedersen, A. K., Peters, G. H., Petersen, A. S., Andersen, H. S., Branner, S., Mortensen, S. B., and Moller, N. P. (2002) J. Biol. Chem. 277, 19982–19990[Abstract/Free Full Text]
  63. Cantrell, D. (1996) Annu. Rev. Immunol. 14, 259–274[CrossRef][Medline] [Order article via Infotrieve]
  64. Mustelin, T., Rahmouni, S., Bottini, N., and Alonso, A. (2003) Immunol. Rev. 191, 139–147[CrossRef][Medline] [Order article via Infotrieve]

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