Winkler, Duane D.

Permanent URI for this collectionhttps://hdl.handle.net/10735.1/5913

Duane Winkler is an Assistant Professor in the Department of Biological Sciences. In 2016, Dr. Winkler received a five-year $1.3 million grant from the National Institute of General Medical Sciences to study the biological mechanisms that underlie Alzheimer's disease and other neurodegenerative disorders in order to develop new treatment strategies.

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    Quantifying the Interaction between Copper-Zinc Superoxide Dismutase (Sod1) and Its Copper Chaperone (Ccs1)
    Boyd, Stefanie D.; Liu, Li; Bulla, Lee A.; Winkler, Duane D.; Boyd, Stefanie D.; Liu, Li; Bulla, Lee A.; Winkler, Duane D.
    Immature copper-zinc superoxide dismutase (Sod1) is activated by its copper chaperone (Ccs1). Ccs1 delivers a single copper ion and catalyzes oxidation of an intra-subunit disulfide bond within each Sod1 monomer through a mechanistically ambiguous process. Here, we use residue specific fluorescent labeling of immature Sod1 to quantitate the thermodynamics of the Sod1Ccs1 interaction while determining a more complete view of Ccs1 function. Ccs1 preferentially binds a completely immature form of Sod1 that is metal deficient and disulfide reduced (E, E-Sod1SH). However, binding induces structural changes that promote high-affinity zinc binding by the Ccs1-bound Sod1 molecule. This adds further support to the notion that Ccs1 likely plays dual chaperoning roles during the Sod1 maturation process. Further analysis reveals that in addition to the copper-dependent roles during Sod1 activation, the N- and C-terminal domains of Ccs1 also have synergistic roles in securing both Sod1 recognition and its own active conformation. These results provide new and measurable analyses of the molecular determinants guiding Ccs1-mediated Sod1 activation.
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    FACT Is a Sensor of DNA Torsional Stress in Eukaryotic Cells
    (2018-09-24) Safina, A.; Cheney, P.; Pal, M.; Brodsky, L.; Ivanov, A.; Kirsanov, K.; Lesovaya, E.; Naberezhnov, D.; Nesher, E.; Koman, I.; Wang, D.; Wang, J.; Yakubovskaya, M.; Winkler, Duane D.; Gurova, K.; Winkler, Duane D.
    Transitions of B-DNA to alternative DNA structures (ADS) can be triggered by negative torsional strain, which occurs during replication and transcription, and may lead to genomic instability. However, how ADS are recognized in cells is unclear. We found that the binding of candidate anticancer drug, curaxin, to cellular DNA results in uncoiling of nucleosomal DNA, accumulation of negative supercoiling and conversion of multiple regions of genomic DNA into left-handed Z-form. Histone chaperone FACT binds rapidly to the same regions via the SSRP1 subunit in curaxin-treated cells. In vitro binding of purified SSRP1 or its isolated CID domain to a methylated DNA fragment containing alternating purine/pyrimidines, which is prone to Z-DNA transition, is much stronger than to other types of DNA. We propose that FACT can recognize and bind Z-DNA or DNA in transition from a B to Z form. Binding of FACT to these genomic regions triggers a p53 response. Furthermore, FACT has been shown to bind to other types of ADS through a different structural domain, which also leads to p53 activation. Thus, we propose that FACT acts as a sensor of ADS formation in cells. Recognition of ADS by FACT followed by a p53 response may explain the role of FACT in DNA damage prevention. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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    Copper-Zinc Superoxide Dismutase Is Activated through a Sulfenic Acid Intermediate at a Copper Ion Entry Site
    (American Society for Biochemistry and Molecular Biology Inc, 2018-08-20) Fetherolf, M. M.; Boyd, Stefanie D.; Taylor, A. B.; Kim, H. J.; Wohlschlegel, J. A.; Blackburn, N. J.; Hart, P. J.; Winge, D. R.; Winkler, Duane D.; Boyd, Stefanie D.; Winkler, Duane D.
    Metallochaperones are a diverse family of trafficking molecules that provide metal ions to protein targets for use as cofactors. The copper chaperone for superoxide dismutase (Ccs1) activates immature copper-zinc superoxide dismutase (Sod1) by delivering copper and facilitating the oxidation of the Sod1 intramolecular disulfide bond. Here, we present structural, spectroscopic, and cell-based data supporting a novel copper-induced mechanism for Sod1 activation. Ccs1 binding exposes an electropositive cavity and proposed “entry site” for copper ion delivery on immature Sod1. Copper-mediated sulfenylation leads to a sulfenic acid intermediate that eventually resolves to form the Sod1 disulfide bond with concomitant release of copper into the Sod1 active site. Sod1 is the predominant disulfide bond-requiring enzyme in the cytoplasm, and this copper-induced mechanism of disulfide bond formation obviates the need for a thiol/disulfide oxidoreductase in that compartment.

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