ABSTRACT
Gold magnetic particles (GMP) are magnetic iron oxide particles modified with gold nanoparticles. The gold particles of GMP specifically bind to cysteine and methionine through Au-S binding. The aim of the present study was to establish a quick and easy protein purification system using novel peptide tags and GMP. Here, we created a variety of peptide tags containing methionine and cysteine and analyzed their affinity to GMP. Binding assays using enhanced green fluorescent protein (EGFP) as a model protein indicated that the tandem methionine tags comprising methionine residues had higher affinity to the GMP than tags comprising both methionine and cysteine residues. Tags comprising both methionine and glycine residues showed slightly higher affinity to GMP and higher elution efficiency than the all-methionine tags. A protein purification assay using phosphorylcholine-treated GMP demonstrated that both a tandem methionine-tagged EGFP and a methionine and glycine-tagged EGFP were specifically purified from a protein mixture with very high efficiency. The efficiency was comparable to that of a histidine-tagged protein purification system. Together, these novel peptide tags, "methionine tags", specifically bind to GMP and can be used for a highly efficient protein purification system.
Subject(s)
Gold/chemistry , Green Fluorescent Proteins/isolation & purification , Magnetics , Metal Nanoparticles/chemistry , Methionine/chemistry , Peptides/chemistry , Electrophoresis, Polyacrylamide Gel , Ferric Compounds/chemistry , Green Fluorescent Proteins/chemistry , Particle Size , Phosphorylcholine/chemistry , Protein Binding , Surface PropertiesABSTRACT
O(6)-Methylguanine and O(6)-chloroethylguanine, which are the primary cytotoxic DNA lesions produced by 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (dacarbazine) and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU), respectively, can be repaired by O(6)-methylguanine-DNA methyltransferase (MGMT), coded by the MGMT gene. However, the two types of drugs exhibit different effects on cells defective in both MGMT and MLH1 functions, the latter being related to the cellular activity to recognize mismatched bases of DNA for inducing apoptosis. Human cells deficient in both MGMT and MLH1 are resistant to the killing effect of dacarbazine and exhibit an increased mutant frequency after treatment with dacarbazine. On the other hand, these doubly deficient cells are sensitive to the killing action of ACNU and there is no significant increase in ACNU-induced mutant frequency. A mismatch recognition complex, composed of MSH2, MSH6, MLH1, PMS2 and PCNA, is formed after exposing MGMT-deficient cells to dacarbazine, but not in cells treated with ACNU. In contrast, the phosphorylation of Chk1 efficiently occurs in cells treated with dacarbazine as well as with ACNU, the former being in MLH1-dependent manner, whereas the latter in MLH1-independent manner. Therefore, the signals delivered from different sources would merge at the step of Chk1 activation or at an earlier step, and the subsequent process leading to apoptosis appears to be common.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Dacarbazine/pharmacology , Nimustine/pharmacology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/metabolism , Checkpoint Kinase 1 , DNA Modification Methylases/genetics , DNA Modification Methylases/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Humans , Mismatch Repair Endonuclease PMS2 , MutL Protein Homolog 1 , MutS Homolog 2 Protein/metabolism , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Protein Kinases/metabolism , Signal Transduction , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolismABSTRACT
Modified bases, such as O6-methylguanines, are produced in cells exposed to alkylating agents and cause apoptosis. In human cells treated with N-methyl-N-nitrosourea, we detected a protein complex composed of MutSalpha, MutLalpha and PCNA on damaged DNA by immunoprecipitation method using chromatin extracts, in which protein-protein interactions were stabilized by chemical crosslinking. Time course experiments revealed that MutSalpha, consisting of MSH2 and MSH6 proteins, and PCNA bind to DNA to form an initial complex, and MutLalpha, composed of MLH1 and PMS2, binds to the complex when the DNA is damaged. This sequential mode of binding was further confirmed by the findings that the association of PCNA-MutSalpha complex on chromatin was observed even in the cells that lack MLH1, whereas in the absence of MSH2 no association of MutLalpha with the chromatin was achieved. Moreover, reduction in the PCNA content by small-interfering RNA or inhibition of DNA replication by aphidicolin, an inhibitor of DNA polymerase, significantly reduced the levels of the PCNA-MutSalpha-MutLalpha complex and also suppressed an increase in the caspase-3 activity, a hallmark for the induction of apoptosis. These observations imply that the induction of apoptosis is coupled with the progression of DNA replication through the action of PCNA.
