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Date of Award
Doctor of Philosophy (PhD)
Michael J. Maroney
Craig T. Martin
Biochemistry | Chemistry | Surgery | Toxicology
The toxicity of nickel compounds is most clearly demonstrated in nickel refinery workers. Nickel exposure leads to lung and nasal cancers and increased risk of acute respiratory syndromes. The toxicity of nickel compounds has been attributed to its roles in oxidative damage, changes in gene expression by an epigenetic mechanism, and inhibition of iron containing enzymes. For this reason, we are interested in the mechanisms of nickel induced inhibition of some of these enzymes. Non-heme iron enzymes carry out a broad number of essential biological reactions in mammalian metabolism, including several that are involved DNA repair and histone demethylation, and thus are involved in gene expression by an epigenetic mechanism. The enzymes involved in DNA repair ( e.g. , ABH2) and histone demethylation ( e.g. , JMJD2 proteins) require Fe(II) and αKG for their function. The replacement of Fe(II) in the active site by other metal ions, including Ni(II), produces an inactive enzyme.
Cellular DNA undergoes alkylation damage by chemicals that modify DNA bases and this damage can be inherited. Cells have evolved systems to repair this DNA alkylation damage. In human, ABH2 repairs endogenously formed 1-methyladenine (1-MeA) and 3-methylcytosine (3-MeC). Our experiment suggests that nickel inhibits ABH2 in a dose dependent manner. The inactivation of ABH2 will lead to increased DNA methylation and subsequent transcriptional silencing, which can result in caner and other developmental defects. Histone tails undergo a number of posttranslational modification including acetylation, methylation, phosphorylation and ubiquitination. Differential methylation of histone H3 and H4 lysyl residues regulates processes including heterochromatin formation, X-chromosome inactivation, DNA repair and transcriptional regulation. The increase in cellular nickel concentration leads to an increase in the global levels of H3K9Me1 and H3K9Me2 - not by affecting histone methyltransferases, but by inhibiting a group of Fe(II) and αKG dependent histone demethylases. Using JMJD2A and JMJD2C as examples, we show that JMJD2 family of histone demethylases is also highly sensitive to inhibition by Ni(II) ions.
Isothermal titration calorimetry (ITC) has been used to understand the binding of Fe(II) and Ni(II) to ABH2. Our ITC results indicate that both Fe(II) and Ni(II) form 1:1 complexes with ABH2, but Ni(II) binds ABH2 stronger than Fe(II). X-ray absorption spectroscopy (XAS) has been used as a structural probe of the active site metal center in WT-recombinant ABH2, truncated JMJD2 proteins (1 - 350 aa) and Ni(II)-substituted WT-recombinant ABH2 and JMD2 proteins in the presence and/or absence of αKG and substrate in order to examine the reaction mechanism and ascertain the intermediate(s) affected by nickel substitution. Our XAS results indicate that in the presence of both αKG and substrate the iron site is five coordinate while the nickel site is six coordinate and thus, nickel site does not have any empty coordination site for oxygen binding and activation.
Thus, using ABH2 and JMJD2 proteins as examples, we show that Fe(II) and αKG dependent enzymes are inhibited by Ni(II) ions. This is consistent with previous reports in literature on other Fe(II) and αKG dependent enzymes. Our results indicate that Ni(II) inhibits ABH2 and JMJD2 proteins by replacing the active site metal and both electronic and steric components are involved. Inhibition of some of these non-heme iron enzymes, like histone demethylases may be one way nickel can alter gene expression by an epigenetic mechanism.
Giri, Nitai Charan, "Structural Investigations Of Early Intremediates And Nickel Inhibitions Complexes Of Human DNA and Histone Demethylases" (2013). Doctoral Dissertations 1896 - February 2014. 464.