Presentation Title

A Novel Role of Histones in the Reduction and Provision of Copper for Mitochondrial Respiration and Superoxide Dismutase Function

Faculty Mentor

Siavash Kurdistani

Start Date

17-11-2018 10:00 AM

End Date

17-11-2018 10:15 AM

Location

C164

Session

Oral 2

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

The eukaryotic genome is packaged by histones that compact the genome and regulate DNA-based processes through histone tails and post-translational modifications. This study investigates a new purpose for the histone H3-H4 tetramer as a copper reductase that affects copper and iron homeostasis. The Kurdistani lab generated a mutation in residue 113, converting histidine to asparagine (H113N). It was observed that H113N defects can be rescued by increasing copper content, suggesting that the function of histone H3 is to optimize copper utilization. Copper is vital for the function of cytochrome c oxidase(Complex IV) in the electron transport chain via the cox17 chaperone protein. Copper is also known to regulate high affinity iron uptake, through the fet3 and fet5 genes in yeast. Copper is needed as well for superoxide dismutase function, sod1. It was hypothesized that histones would thus regulate copper and iron homeostasis by controlling the copper levels used for Complex IV, iron uptake, and sod1 function. Oxygen consumption assays showed the H113N mutation had reduced respiration that was recovered by 50 µM CuSO₄. Also, the H113N mutation increased sensitivity to iron depletion, induced by BPS, an iron chelator, or by an alkaline environment in growth assays. Using PCR-mediated gene deletion, it was discovered that the defect in iron homeostasis is not due to fet3 or fet5. Further experimentation proved that sod1 is responsible for the defection in iron homeostasis for histones, because addition of 1 μM Cu recovered H113N in the various sod1 deletion backgrounds. Through inductively coupled plasma mass spectrometry, it was confirmed that H113N has similar levels of copper and iron, supporting the idea that the defect is due to sod1 dysfunction. This study helps determine the unprecedented idea that histones provide reduced copper to the cell and has implications in both copper and iron homeostasis.

Summary of research results to be presented

In vitro studies show activity and reduction of copper with the histone H3-H4 tetramer and decreased reduction in H113N tetramers. Where is the reduced copper going? Oxygen consumption assays show that the H113N mutation has 50% less mitochondrial respiration in comparison to wild type in respiration-induced media with 50 µM CuSO₄. This defect in mitochondrial respiration is recovered with 150 µM CuSO₄ to 95% of wild type oxygen consumption levels. Also, the addition of antimycin A, which inhibits the electron transport chain, abolishes the oxygen consumption defect in H113N. Mutation of the histone 110 residue to a cysteine (A110C) is thought to be beneficial for the enzymatic properties of the copper reductase tetramer due to the chelating and electron transferring properties of cysteine. Oxygen consumption assays of the A110C mutation show a gain of function to 145% of wild type levels. The defect in oxygen consumption in wild type is recovered by copper. These results point to the idea that the H3-H4 tetramer provides copper for the electron transport chain. Also, H113N has growth defects in iron limiting conditions due to the overlaps between copper and iron homeostasis. Iron uptake measurements through inductively coupled plasma mass spectrometry show that the total amount of iron within the cell is the same between wild type and H113N. Growth assays show that the difference between H113N and wild type abolishes with the deletion of sod1. These results point to the idea that histones affect iron homeostasis by providing copper to sod1.

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Nov 17th, 10:00 AM Nov 17th, 10:15 AM

A Novel Role of Histones in the Reduction and Provision of Copper for Mitochondrial Respiration and Superoxide Dismutase Function

C164

The eukaryotic genome is packaged by histones that compact the genome and regulate DNA-based processes through histone tails and post-translational modifications. This study investigates a new purpose for the histone H3-H4 tetramer as a copper reductase that affects copper and iron homeostasis. The Kurdistani lab generated a mutation in residue 113, converting histidine to asparagine (H113N). It was observed that H113N defects can be rescued by increasing copper content, suggesting that the function of histone H3 is to optimize copper utilization. Copper is vital for the function of cytochrome c oxidase(Complex IV) in the electron transport chain via the cox17 chaperone protein. Copper is also known to regulate high affinity iron uptake, through the fet3 and fet5 genes in yeast. Copper is needed as well for superoxide dismutase function, sod1. It was hypothesized that histones would thus regulate copper and iron homeostasis by controlling the copper levels used for Complex IV, iron uptake, and sod1 function. Oxygen consumption assays showed the H113N mutation had reduced respiration that was recovered by 50 µM CuSO₄. Also, the H113N mutation increased sensitivity to iron depletion, induced by BPS, an iron chelator, or by an alkaline environment in growth assays. Using PCR-mediated gene deletion, it was discovered that the defect in iron homeostasis is not due to fet3 or fet5. Further experimentation proved that sod1 is responsible for the defection in iron homeostasis for histones, because addition of 1 μM Cu recovered H113N in the various sod1 deletion backgrounds. Through inductively coupled plasma mass spectrometry, it was confirmed that H113N has similar levels of copper and iron, supporting the idea that the defect is due to sod1 dysfunction. This study helps determine the unprecedented idea that histones provide reduced copper to the cell and has implications in both copper and iron homeostasis.