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Comparative Characterization of the Major Human Glutaredoxin Isozymes and Identification of a Mechanism by which Grx1 Regulates Apoptosis in Cardiomyocytes

Gallogly, Molly Megan
http://rave.ohiolink.edu/etdc/view?acc_num=case1247255429

Abstract Details

2009, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Mixed disulfides between cysteines and glutathione (GSH) (i.e., protein S-glutathionylation) represent an oxidative post-translational modification with roles in sulfhydryl homeostasis and signal transduction. Glutaredoxin (Grx) is an efficient catalyst of protein deglutathionylation, and human Grx1 (cytosolic isoform) is the prototype enzyme. Recently, a second isoform (Grx2) was discovered. Systematic analysis of the human glutaredoxins demonstrated remarkable similarities, including specificity for glutathionyl mixed disulfides and double displacement, encounter-type catalysis where reduction of the Grx-SSG intermediate is rate-determining. Both Grx1 and Grx2 are recycled preferentially by GSH, and both promote protein glutathionylation with activated donors glutathione thiyl radical and GSNO. However, Grx2 has 10-fold lower catalytic efficiency than Grx1, due to decreased Vmax. Lower rate enhancement by Grx2 is explained by (a) 1 pH unit increase in pKa of the catalytic cysteine serving as leaving group in the rate-determining step; and (b) less enhancement of GSH nucleophilicity towards the Grx-SSG intermediate. Calculated concentrations of Grx1 and Grx2 suggest equal deglutathionylase activities for the subcompartments of mitochondria, and potentially more glutathione thiyl radical-mediated glutathionylation by Grx2 in the matrix. Redox regulation of apoptosis is an area of intense study. We examined Grx1 regulation of apoptosis in cardiomyocytes, which exhibit increased apoptotic susceptibility and diminished Grx1 with aging in animal models. To determine whether Grx1 deficiency increases apoptotic susceptibility, Grx1 was knocked down in H9c2 cardiomyocytes to a similar extent as in cardiomyocytes from elderly animals. Grx1 knock-down resulted in increased apoptosis at baseline and following oxidative stress; concomitantly NFκB transcriptional activity was diminished, and its anti-apoptotic gene products Bcl-2 and Bcl-xL were decreased. Inhibiting NFκB, or knocking down Bcl-2 and/or Bcl-xL in wild-type H9c2 cells to the same extent as in Grx1 knock-down cells increased apoptosis at baseline; but only Bcl-xL knock-down increased oxidant-induced apoptosis and fully accounted for the apoptotic phenotype observed in Grx1-deficient cells. NFκB activity was diminished also in cardiomyocytes from elderly Fischer 344 rats, indicating correspondence of the cellular and animal models of aging. This thesis provides foundations for future investigations on: (a) specific roles of glutaredoxins in sulfhydryl regulation within mitochondria; and (b) mechanisms of Grx1-dependent regulation of apoptotic signaling in cardiomyocytes.
John Meiyal, PhD (Advisor)
Charles Hoppel, MD (Committee Chair)
Clark Distelhorst, MD (Committee Member)
George Dubyak, PhD (Committee Member)
Michael Maguire, PhD (Committee Member)
314 p.
Pharmacology
glutaredoxin; glutathionylation; oxidative stress; redox signaling; apoptosis; Bcl-xL; Bcl-2; cardiomyocyte

Recommended Citations

Citations

  • Gallogly, M. M. (2009). Comparative Characterization of the Major Human Glutaredoxin Isozymes and Identification of a Mechanism by which Grx1 Regulates Apoptosis in Cardiomyocytes [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1247255429

    APA Style (7th edition)

  • Gallogly, Molly. Comparative Characterization of the Major Human Glutaredoxin Isozymes and Identification of a Mechanism by which Grx1 Regulates Apoptosis in Cardiomyocytes. 2009. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1247255429.

    MLA Style (8th edition)

  • Gallogly, Molly. "Comparative Characterization of the Major Human Glutaredoxin Isozymes and Identification of a Mechanism by which Grx1 Regulates Apoptosis in Cardiomyocytes." Doctoral dissertation, Case Western Reserve University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1247255429

    Chicago Manual of Style (17th edition)

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© 2009, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.