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Title: Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry

Abstract

Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.

Authors:
 [1];  [2];  [1];  [1];  [3]
  1. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106 (United States)
  2. Mass Spectrometry Center, Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195 (United States)
  3. Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, Amarillo, TX 79106 (United States). E-mail: Kalkunte.srivenugopal@ttuhsc.edu
Publication Date:
OSTI Identifier:
20713461
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 337; Journal Issue: 4; Other Information: DOI: 10.1016/j.bbrc.2005.09.177; PII: S0006-291X(05)02219-9; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AFFINITY; ALKYLATION; CELL CYCLE; CHEMOTHERAPY; CHROMATOGRAPHY; DNA DAMAGES; DNA REPAIR; DNA REPLICATION; HEAT-SHOCK PROTEINS; HISTONES; LARGE INTESTINE; MASS SPECTROSCOPY; NEOPLASMS; RNA PROCESSING

Citation Formats

Niture, Suryakant K., Doneanu, Catalin E., Velu, Chinavenmeni S., Bailey, Nathan I., and Srivenugopal, Kalkunte S.. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry. United States: N. p., 2005. Web. doi:10.1016/j.bbrc.2005.09.177.
Niture, Suryakant K., Doneanu, Catalin E., Velu, Chinavenmeni S., Bailey, Nathan I., & Srivenugopal, Kalkunte S.. Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry. United States. doi:10.1016/j.bbrc.2005.09.177.
Niture, Suryakant K., Doneanu, Catalin E., Velu, Chinavenmeni S., Bailey, Nathan I., and Srivenugopal, Kalkunte S.. Fri . "Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry". United States. doi:10.1016/j.bbrc.2005.09.177.
@article{osti_20713461,
title = {Proteomic analysis of human O {sup 6}-methylguanine-DNA methyltransferase by affinity chromatography and tandem mass spectrometry},
author = {Niture, Suryakant K. and Doneanu, Catalin E. and Velu, Chinavenmeni S. and Bailey, Nathan I. and Srivenugopal, Kalkunte S.},
abstractNote = {Recent evidence suggests that human O {sup 6}-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that protects the genome against mutagens and accords tumor resistance to many anticancer alkylating agents, may have other roles besides repair. Therefore, we isolated MGMT-interacting proteins from extracts of HT29 human colon cancer cells using affinity chromatography on MGMT-Sepharose. Specific proteins bound to this column were identified by electrospray ionization tandem mass spectrometry and/or Western blotting. These procedures identified >60 MGMT-interacting proteins with diverse functions including those involved in DNA replication and repair (MCM2, PCNA, ORC1, DNA polymerase {delta}, MSH-2, and DNA-dependent protein kinase), cell cycle progression (CDK1, cyclin B, CDK2, CDC7, CDC10, 14-3-3 protein, and p21{sup waf1/cip1}), RNA processing and translation (poly(A)-binding protein, nucleolin, heterogeneous nuclear ribonucleoproteins, A2/B1, and elongation factor-1{alpha}), several histones (H4, H3.4, and H2A.1), and topoisomerase I. The heat shock proteins, HSP-90{alpha} and {beta}, also bound strongly with MGMT. The DNA repair activity of MGMT was greatly enhanced in the presence of interacting proteins or histones. These data, for the first time, suggest that human MGMT is likely to have additional functions, possibly, in sensing and integrating the DNA damage/repair-related signals with replication, cell cycle progression, and genomic stability.},
doi = {10.1016/j.bbrc.2005.09.177},
journal = {Biochemical and Biophysical Research Communications},
number = 4,
volume = 337,
place = {United States},
year = {Fri Dec 02 00:00:00 EST 2005},
month = {Fri Dec 02 00:00:00 EST 2005}
}
  • A mouse cDNA clone encoding O{sup 6}-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O{sup 6}-alkylguanine in DNA, was cloned from a {lambda}gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction basedmore » on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.« less
  • E. coli Ada protein (39kD) and its 19kD cleavage product act as DNA-O/sup 6/-methylguanine methyltransferase (MGMT) by stoichiometrically accepting the methyl group from promutagenic O/sup 6/-methylguanine residues (and phosphotriesters in the case of Ada protein) in DNA. Methylated Ada acts as a positive regulator for its own gene and some other DNA repair genes. The methyl group of 0/sup 6/-methylguanine is transferred to a specific cysteine residue (in a Pro-Cys-His sequence) in MGMT. The authors have substituted the cysteine with histidine in the Ada protein by oligodeoxynucleotide-directed mutagenesis of the cloned ada gene. Both wild type and an ada (noninducible)more » mutant of E. coli harboring plasmids containing either normal or mutant ada genes have comparable levels of MGMT. Furthermore, the level of induction of MGMT by N-methyl-N'-nitro-N-nitrosoguanidine is similar in all cases. Whether the mutant protein causes induction of host MGMT or is itself active as a methyl acceptor is being investigated. Other MGMT mutants in which the cysteine residue was replaced with alanine or aspartic acid or in which the cysteine-histidine sequence was transposed have been prepared, and their enzymatic activity and autoregulation are being studied.« less
  • O/sup 6/-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing /sup 3/H-labeled O/sup 6/-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not inmore » the structural gene for the methyltransferase.« less
  • A synthetic DNA substrate containing O/sup 6/-methyl(8-/sup 3/H)-guanine was used to assay demethylation of the premutagenic base by O/sup 6/-methylguanine-DNA methyltransferase in extracts of HeLa cells, Chinese hamster ovary cells and normal rat kidney cells which had been treated with multiple doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). No induction of methyltransferase activity was observed in any of the cell lines tested. Constitutive levels of methyltransferase in cell lines proficient (Mex/sup +/) in O/sup 6/-methylguanine repair were decreased in a dose-dependent fashion by either single or multiple treatments with MNNG over a broad range of dose levels. Recovery of constitutive levels of activitymore » required 24- to 48-h incubation periods. Repair deficient (Mex/sup -/) cell lines lacked both constitutive and inducible methyltransferase activity. 42 references, 4 figures, 1 table.« less
  • Alkylating agents play an important role in the chemotherapy of malignant melanomas. The activity of alkylating agents depends on their capacity to form alkyl adducts with DNA, in some cases causing cross-linking of DNA strands. However, the use of these agents is limited by cellular resistance induced by the DNA repair enzyme O{sup 6}-methylguanine DNA-methyltransferase (MGMT) which removes alkyl groups from alkylated DNA strands. To determine to what extent the expression of MGMT in melanoma cells induces resistance to alkylating agents, the human cell line CAL77 Mer- (i.e., MGMT deficient) were transfected with pcMGMT vector containing human MGMT cDNA. Severalmore » clones expressing MGMT at a high level were selected to determine their sensitivity to chemotherapeutic drugs. Melanoma-transfected cells were found to be significantly less sensitive to nitrosoureas (carmustine, fotemustine, streptozotocin) and temozolomide with an increase of IC{sub 5} values between 3 and 14 when compared to parent cells. No difference in cell survival rates between MGMT-proficient and -deficient cells was observed for melphalan, chlorambucil, busulphan, thiotepa and cisplatin which preferentially induce N{sup 7} guanine lesions. Surprisingly, MGMT overexpression increased the sensitivity of CAL77 cells to mitomycin C by approximately 10-fold. Treatment of clonal cell lines with buthionine-[S,R]-sulfoximine (BSO), an inhibitor of {gamma}-glutamylcysteine synthetase which depletes cellular glutathione, completely reversed this unexpected increase in sensitivity to mitomycin C. This observation suggests that glutathione is involved in the sensitivity of MGMT-transfected cells to mitomycin C and may act synergistically with MGMT via an unknown mechanism.« less