Chemical knockdown of protein-tyrosine phosphatase 1B by 1,2-naphthoquinone through covalent modification causes persistent transactivation of epidermal growth factor receptor

1,2-Naphthoquinone (1,2-NQ), an atmospheric contaminant, causes the contraction of guinea pig trachea through the activation of epidermal growth factor receptor (EGFR) by inhibiting protein-tyrosine phosphatases (PTPs). Phosphorylation of EGFR is negatively regulated by PTPs, but details of the mechanism by which 1,2-NQ inhibits PTPs have not been elucidated. Results described in this report demonstrate that 1,2-NQ forms covalent bonds with PTP1B after exposure to human epithelial A431 cells. In this study, a concentration-dependent phosphorylation of EGFR was found to be coupled to the reduction of PTP activity in the cells. The reduction in PTP activity was due to the irreversible modification of PTP1B, and when PTP1B was overexpressed by the cells, the 1,2-NQ-mediated EGFR phosphorylation was suppressed. Studies with purified PTP1B and 1,2-NQ showed that the reduction in enzyme activity was due to a nucleophilic attack by the quinone on the enzyme, to form covalent bonds. Matrix-assisted laser desorption and ionization time-of-flight mass spectrometry analysis and mutation experiments revealed that PTP1B inactivation was primarily due to covalent attachment of the quinone to Cys-121 of the enzyme, with binding to His-25 and Cys-215 as well. Collectively, the results show that covalent attachment of 1,2-NQ to PTP1B is at least partially responsible for the reduction of PTP activity, which leads to prolonged transactivation of EGFR in the cells.     

Characterization of the DNA binding properties of polyomavirus capsid protein.

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.     

Glyceraldehyde-3-phosphate dehydrogenase as a quinone reductase in the suppression of 1,2-naphthoquinone protein adduct formation

1,2-Naphthoquinone (1,2-NQ) is electrophilic, and forms covalent bonds with protein thiols, but its two-electron reduction product 1,2-dihydroxynaphthalene (1,2-NQH(2)) is not, so enzymes catalyzing the reduction with reduced pyridine nucleotides as cofactors could protect cells from electrophile-based chemical insults. To assess this possibility, we examined proteins isolated from the 9000g supernatant from mouse liver for 1,2-NQ reductase activity using an HPLC assay procedure for the hydroquinone of 1,2-NQ and Cibacron Blue 3GA column chromatography and Western blot analysis with specific antibody to determine 1,2-NQ-bound proteins. Among the proteins with high affinities for pyridine nucleotides that also inhibited 1,2-NQ-protein adduct formation in the presence of NADH, a 37-kDa protein was found and identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using recombinant human GAPDH, we found that this glycolytic enzyme indeed catalyzes the two-electron reduction of 1,2-NQ accompanied by extensive NADH consumption under 20% oxygen conditions. When either 1,2-NQH(2) or 1,2-NQ was incubated with GAPDH in the presence of NADH, minimal covalent bonding to the enzyme occurred compared to that in its absence. These results indicate that GAPDH can inhibit 1,2-NQ-based electrophilic protein modification by conversion to the nonelectrophilic 1,2-NQH(2) via an NADH-dependent process.     

Molecular basis of enzyme inactivation by an endogenous electrophile 4-hydroxy-2-nonenal: identification of modification sites in glyceraldehyde-3-phosphate dehydrogenase

4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation-derived reactive aldehyde, is a potent inhibitor of sulfhydryl enzymes, such as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It has been suggested that HNE exerts an inhibitory effect on the enzyme due to the modification of the cysteine residue (Cys-149) at the catalytic site generating the HNE-cysteine Michael addition-type adduct [Uchida, K., and Stadtman, E. R. (1993) J. Biol. Chem. 268, 6388-6393]. In the study presented here, to elucidate the mechanism for the inactivation of GAPDH by HNE, we attempted to identify the modification sites of the enzyme by monitoring the formation of the HNE Michael adducts by mass spectrometric methods. Incubation of GAPDH (1 mg/mL) with 1 mM HNE in 50 mM sodium phosphate buffer (pH 7.4) at 37 degrees C resulted in a time-dependent loss of enzyme activity, which was associated with the covalent binding of HNE to the enzyme. To identify the site of modification of GAPDH by HNE, both the HNE-pretreated and untreated GAPDH were digested with trypsin and V8 protease, and the resulting peptides were subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS). This technique identified five peptides, which contained the HNE adducts at His-164, Cys-244, Cys-281, His-327, and Lys-331 and revealed that both His-164 and Cys-281 were very rapidly modified at 5 min, followed by Cys-244 at 15 min and His-327 and Lys-331 at 30 min. These observations and the observation that the HNE modification of the catalytic center, Cys-149, was not observed suggest that the HNE inactivation of GAPDH is not due to the modification of the catalytic center but to the selective modification of amino acids primarily located in the surface of the GAPDH molecule.     

Amino acids of the Sulfolobus solfataricus mini-chromosome maintenance-like DNA helicase involved in DNA binding/remodeling

Herein we report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. The crystallographic structure of the N-terminal portion (residues 2-286) of the Methanothermobacter thermoautotrophicum MCM protein revealed a dodecameric assembly with two hexameric rings in a head-to-head configuration and a positively charged central channel proposed to encircle DNA molecules. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity. These results have important implications for the understanding of the molecular mechanism of the MCM DNA helicase action.     

Site-specific chemical modification of interleukin-1 beta by acrylodan at cysteine 8 and lysine 103.

Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate the probe on the modified proteins. Both modified proteins carried one molecule of acrylodan each, one at Cys-8 and the other at Lys-103. Neither Cys-71 nor any of the other 13 lysine residues of rIL-1 beta was modified. Cysteine 71 is inaccessible to acrylodan, but the unusual specificity for Lys-103 could be caused by the location of that residue at the bottom of a hydrophobic pocket which might specifically bind the reagent. No double-labeled protein was detected, indicating that the introduction of the label at either site interferes with the labeling at the other. Both acrylodan-modified proteins exhibited bioactivity in the thymocyte proliferation assay at a level equivalent to that of the unmodified control protein (1.7 x 10(7) units/mg), which shows that the modification of either the Cys-8 or Lys-103 position with acrylodan does not interfere with the cellular bioactivities of the respective proteins. Furthermore, receptor binding assays yielded a Kd = 32.0 +/- 4.8 pM for the Lys-103-labeled protein, Kd = 69.5 +/- 12.7 pM for the unmodified protein, and Kd = 75.0 +/- 11.6 pM for the Cys-8-labeled protein. Thus, Cys-8 or Lys-103 modification of rIL-1 beta by acrylodan also does not interfere with the ability of the molecule to bind to its receptor. The slightly higher affinity of the Lys-103-labeled protein for the receptor suggests that the positive charge on this residue in the native molecule may interfere with IL-1 receptor binding. The two fluorescent labeled IL-1 proteins described herein should provide interesting probes for the study of IL-1/IL-1 receptor interactions.     

Differential inhibition of Arabidopsis methionine adenosyltransferases by protein S-nitrosylation

In animals, protein S-nitrosylation, the covalent attachment of NO to the thiol group of cysteine residues, is an intensively investigated posttranslational modification, which regulates many different processes. A growing body of evidence suggests that this type of redox-based regulation mechanism plays a pivotal role in plants, too. Here we report the molecular mechanism for S-nitrosylation of methionine adenosyltransferase (MAT) of Arabidopsis thaliana, thereby presenting the first detailed characterization of S-nitrosylation in plants. We cloned three MAT isoforms of Arabidopsis and tested the effect of NO on the activity of the purified, recombinant proteins. Our data showed that incubation with GSNO resulted in blunt, reversible inhibition of MAT1, whereas MAT2 and MAT3 were not significantly affected. Cys-114 of MAT1 was identified as the most promising target of NO-induced inhibition of MAT1, because this residue is absent in MAT2 and MAT3. Structural analysis of MAT1 revealed that Cys-114 is located nearby the putative substrate binding site of this enzyme. Furthermore, Cys-114 is flanked by S-nitrosylation-promoting amino acids. The inhibitory effect of GSNO was drastically reduced when Cys-114 of MAT1 was replaced by arginine, and mass spectrometric analyses of Cys-114-containing peptides obtained after chymotryptic digestion demonstrated that Cys-114 of MAT1 is indeed S-nitrosylated. Because MAT catalyzes the synthesis of the ethylene precursor S-adenosylmethionine and NO is known to influence ethylene production in plants, this enzyme probably mediates the cross-talk between ethylene and NO signaling.     

Identification of functionally relevant residues of the rat ileal apical sodium-dependent bile acid cotransporter

The mechanisms underlying the transport of bile acids by apical sodium-dependent bile acid transporter (Asbt) are not well defined. To further identify the functionally relevant residues, thirteen conserved negatively (Asp and Glu) and positively (Lys and Arg) charged residues plus Cys-270 of rat Asbt were replaced with Ala or Gln by site-directed mutagenesis. Seven of the fourteen residues of rat Asbt were identified as functionally important by taurocholate transport studies, substrate inhibition assays, confocal microscopy, and electrophysiological methods. The results showed that Asp-122, Lys-191, Lys-225, Lys-256, Glu-261, and Lys-312,Lys-313 residues of rat Asbt are critical for transport function and may determine substrate specificity. Arg-64 may be located at a different binding site to assist in interaction with non-bile acid organic anions. For bile acid transport by Asbt, Na(+) ion movement is a voltage-dependent process that tightly companied with taurocholate movement. Asp-122 and Glu-261 play a critical role in the interaction of a Na(+) ion and ligand with Asbt. Cys-270 is not essential for the transport process. These studies provide new details about the amino acid residues of Asbt involved in binding and transport of bile acids and Na(+).     

Site-directed mutagenesis of the FAD-binding histidine of 6-hydroxy-D-nicotine oxidase. Consequences on flavinylation and enzyme activity

In 6-hydroxy-D-nicotine oxidase (6-HDNO) FAD is covalently bound to His71 of the polypeptide chain by an 8 alpha-(N3-histidyl)-riboflavin linkage. The FAD-binding histidine was exchanged by site-directed mutagenesis to either a Cys- or Tyr-residue, two amino acids known to be involved in covalent binding of FAD in other enzymes, or to a Ser-residue. None of the amino acid replacements for His71 allowed covalent FAD incorporation into the 6-HDNO polypeptide. Thus, the amino acid residues involved in covalent FAD-binding require a specific polypeptide surrounding in order for this modification to proceed and cannot be replaced with each other. Enzyme activity was completely abolished with Tyr in place of His71. 6-HDNO activity with non-covalently bound FAD was found with 6-HDNO-Cys and to a lesser extent also with 6-HDNO-Ser. However, the Km values for 6-HDNO-Cys and 6-HDNO-Ser were increased approximately 20-fold as compared to 6-HDNO-His. Both mutant enzymes, in contrast to the wild-type enzyme, needed additional FAD in the enzymatic assay (50 microM for 6-HDNO-Ser and 10 microM for 6-HDNO-Cys) for maximal enzyme activity.     

Catalytic mechanism of DNA topoisomerase IB

Type IB topoisomerases and tyrosine recombinases are structurally homologous strand transferases that act through DNA-(3'-phosphotyrosyl)-enzyme intermediates. A constellation of conserved amino acids (Arg-130, Lys-167, Arg-223, and His-265 in vaccinia topoisomerase) catalyzes transesterification of tyrosine to the scissile phosphodiester. We used 5'-bridging phosphorothiolate-modified DNAs to implicate Lys-167 as a general acid catalyst. The lower pKa of the 5'-S leaving group versus 5'-O restored activity to the K167A mutant, whereas there was no positive thio effect for mutants R223A and H265A. The lysine is located atop a flexible hairpin loop, and it shifts into the minor groove upon DNA binding. Coupling of conformational changes in a general acid loop to covalent catalysis of phosphoryl transfer is one of several mechanistic features shared by the topoisomerase/recombinase and protein phosphatase superfamilies.     

Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine

We have labeled the adenosine triphosphate binding site of Escherichia coli DNA gyrase with the ATP affinity analog, [3H]pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP). PLP-AMP strongly inhibits the ATP-ase and DNA supercoiling activities of DNA gyrase, with 50% inhibition occurring at 7.5 microM inhibitor. ATP and ADP compete with PLP-AMP for binding and protect the enzyme against inhibition. The labeling appears to proceed by a Schiff base complex between the 4-formyl group of the pyridoxyl moiety of PLP-AMP and a protein primary amino group, since the inhibition and reagent labeling are reversible unless the complex is treated with NaBH4. Complete inactivation is estimated to occur upon the covalent incorporation of 2 mol of inhibitor/mol of gyrase. The Km for ATP was found to be unchanged for partially inhibited enzyme samples, suggesting an all-or-none type of inhibition. A 3H-labeled peptide spanning residues 93-131 of the B protein was isolated from a V-8 protease digest. Radioactive peaks corresponding to Lys-103 and Lys-110 were found during the Edman degradation, suggesting that these amino acids form part of the ATP binding site. A comparison of the amino acid sequence in this region with the sequences of other type II topoisomerases indicates the possible location of a common ATP binding domain.     

ATPase, GTPase, and RNA binding activities associated with the 206-kilodalton protein of turnip yellow mosaic virus.

The 206-kDa protein of turnip yellow mosaic virus belongs to an expanding group of proteins containing a domain which includes the consensus nucleotide binding site GxxxxGKS/T. A portion of this protein (amino acids [aa] 916 to 1259) was expressed in Escherichia coli and purified by affinity chromatography to near homogeneity. In the absence of any other viral factors, it exhibited ATPase and GTPase activities in vitro. A mutant protein with a single amino acid substitution in the consensus nucleotide binding site (Lys-982 to Ser) exhibited only low levels of both activities, implying that Lys-982 is important for nucleoside triphosphatase activity. The protein also possessed nonspecific RNA binding capacity. Deletion mutants revealed that an N-terminal domain (aa 916 to 1061) and a C-terminal domain (aa 1182 to 1259) participate in RNA binding. The results presented here provide the first experimental evidence that turnip yellow mosaic virus encodes nucleoside triphosphatase and RNA binding activities.     

The lysine residues implicated in the gene 5 protein association sites

Gene 5 protein, a DNA unwinding protein encoded by the bacteriophage fd, is self-associative in presence of DNA or oligonucleotides. The lysine residues implicated in the protein-protein binding domains have been identified after modification with acetimidate by means of peptide and amino acid analyses. These residues are Lys-7 and Lys-69.     

The structure of jack bean urease. The complete amino acid sequence, limited proteolysis and reactive cysteine residues

The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90,770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickel-binding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59, 207, 592, 663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.     

Properties of the DNA-binding domain of the simian virus 40 large T antigen.

T antigen (Tag) from simian virus 40 binds specifically to two distinct sites in the viral origin of replication and to single-stranded DNA. Analysis of the protein domain responsible for these activities revealed the following. (i) The C-terminal boundary of the origin-specific and single-strand-specific DNA-binding domain is at or near amino acid 246; furthermore, the maximum of these DNA-binding activities coincides with a narrow C-terminal boundary, spanning 4 amino acids (246 to 249) and declines sharply in proteins with C termini which differ by a few (4 to 10) amino acids; (ii) a polypeptide spanning residues 132 to 246 of Tag is an independent domain responsible for origin-specific DNA binding and presumably for single-stranded DNA binding; and (iii) a comparison of identical N-terminal fragments of Tag purified from mammalian and bacterial cells revealed differential specificity and levels of activity between the two sources of protein. A role for posttranslational modification (phosphorylation) in controlling the DNA-binding activity of Tag is discussed.